Abstract
Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.
Highlights
Natural human glutathione peroxidase 1 is an important antioxidant enzyme that can protect the body from oxidative damage by catalyzing the reduction of hydroperoxides using glutathione (GSH) as a reductant [1]
We prepared a high yield and catalytic activity selenium-dependent glutathione peroxidase 1 mutant (GPx1M) by mutating cysteine (C 78, C 115, and C 156) in human glutathione peroxidase 1 (hGPx1) to serine in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system [4]
Based on the structure-function relationships between glutathione and GPx1M, Lys 88 and Lys 166 are near the GPx1M catalytic active center (Sec 49, Gln 84, and Trp 162), which might reduce its enzymatic activity
Summary
Natural human glutathione peroxidase 1 (hGPx1) is an important antioxidant enzyme that can protect the body from oxidative damage by catalyzing the reduction of hydroperoxides using glutathione (GSH) as a reductant [1]. The use of hGPx1 is affected by its limited sources and poor stability [2, 3] To solve this problem, we prepared a high yield and catalytic activity selenium-dependent glutathione peroxidase 1 mutant (GPx1M) by mutating cysteine (C 78, C 115, and C 156) in hGPx1 to serine in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system [4]. As with many other therapeutic peptides and proteins, GPx1 has a low stability and short plasma half-life [5]. These shortcomings limit the clinical application of GPx1 and its mutant protein
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