新型跨膜融合抗氧化酶的构建及其对过氧化氢诱导细胞氧化损伤的保护作用

Construction of novel transmembrane fusion antioxidant enzymes and their protective effect against hydrogen peroxide-mediated cellular oxidative damage

摘要:

活性氧(reactive oxygen species,ROS)是放射治疗引发肿瘤患者副作用的重要介导因素之一。由谷胱甘肽S-转移酶(glutathione S-transferase,GST)、超氧化物歧化酶1(superoxide dismutase 1,SOD1)和跨膜肽构成的融合抗氧化酶可有效抵御ROS损伤。将2种抗氧化酶进一步与基质金属蛋白酶-2/9底物X肽及跨膜肽R9融合后，得到的融合抗氧化酶GST-SOD1-X-R9(GS1XR)跨膜进入肿瘤细胞的能力降低，从而实现对正常细胞的靶向防护。然而，非人源GST的应用可能具有免疫原性风险，为解决这一问题，本研究利用无缝克隆技术构建含人源GST基因的表达载体，用于非人源GST基因的替换，进而表达和纯化了新型跨膜融合抗氧化酶GS1R和GS1XR。随后，以GS1为对照，评估新型GS1R和GS1XR对H₂O₂诱导的L-02细胞氧化损伤的保护作用。实验结果表明，新型GS1XR的GST比活性与改造前蛋白保持一致，但SOD比活性有所提高。200 μmol/L H₂O₂可短暂激活核因子-红细胞2-相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)通路，然而，24 h后激活效应减弱，导致细胞活力下降至48.4%。GS1R和GS1XR可清除胞内ROS，直接抵御氧化损伤，并促进Nrf2入核激活抗氧化通路，使细胞活力恢复至正常水平，且两者保护作用相当。相比之下，缺乏跨膜能力的GS1仅能清除胞外ROS，保护效果有限。综上所述，2种新型融合抗氧化酶在抵御ROS介导的正常细胞氧化损伤方面均展现出良好的应用潜力，本研究为其在相关领域的进一步研究奠定了基础。

Abstract:

Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.