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

Jianru PAN; Ziyi ZHANG; Jinnan CHU; Yanan HAN; Xueying ZHENG; Shirong CAI; Huocong HE

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

Author: Jianru PAN ¹ ; Ziyi ZHANG ¹ ; Jinnan CHU ¹ ; Yanan HAN ¹ ; Xueying ZHENG ¹ ; Shirong CAI ¹ ; Huocong HE ²
Author Information

1. College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, Fujian, China.
2. Laboratory of Radiation Biology and Oncology, Fujian Medical University Cancer Hospital & Fujian Cancer Hospital, Fuzhou 350014, Fujian, China.
Publication Type:Journal Article
Keywords: fusion antioxidant enzymes; hepatocyte; hydrogen peroxide (H2O2); oxidative damage
MeSH: Humans; Oxidative Stress/drug effects*; Hydrogen Peroxide; Antioxidants/metabolism*; Glutathione Transferase/metabolism*; Recombinant Fusion Proteins/pharmacology*; Superoxide Dismutase-1; Reactive Oxygen Species/metabolism*; Superoxide Dismutase/biosynthesis*
From: Chinese Journal of Biotechnology 2025;41(4):1547-1558
CountryChina
Language:Chinese
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.