Construction and activity analyses of single functional mouse peroxiredoxin 6 (Prdx6)

Lu-Lu Wang 1 , Shi-Ying Lu 1 , Pan Hu 1 , Bao-Quan Fu 2 , Yan-Song Li 1 , Fei-Fei Zhai 1 , Dan-Di Ju 1 , Shi-Jun Zhang 1 , Bing Su 1 , Yu Zhou 1 , Zeng-Shan Liu 1 , and Hong-Lin Ren 1
  • 1 Key Laboratory of Zoonosis Research, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, 130062, Changchun, China
  • 2 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of the Ministry of Agriculture, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 730046, Lanzhou, China


Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection.

Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined.

Results: The core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit.

Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

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  • 1. Benipal B., Feinstein S.I., Chatterjee S., Dodia C., Fisher A.B.: Inhibition of the phospholipase A2 activity of peroxiredoxin 6 prevents lung damage with exposure to hyperoxia. Redox Biol 2015, 4, 321–327.

  • 2. Chatterjee S., Feinstein S.I., Dodia C., Sorokina E., Lien Y.C., Nguyen S., Debolt K., Speicher D., Fisher A.B.: Peroxiredoxin 6, phosphorylation, and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages. J Biol Chem 2011, 286, 11696–11706.

  • 3. Chhunchha B., Kubo E., Fatma N., Singh D.P.: Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation. Cell Death Dis 2017, 8, 2525–2525.

  • 4. Fisher A.B.: Peroxiredoxin 6: a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities. Antioxid Redox Signal 2011, 15, 831–844.

  • 5. Fisher A.B., Dodia C.: Role of acidic Ca2+-independent phospholipase A2 in synthesis of lung dipalmitoyl phosphatidylcholine. Am J Physiol-Lung Cell Molec Physiol 1997, 272, 238–243.

  • 6. Fisher A.B., Dodia C., Feinstein S.I., Ho Y.S.: Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2. J Lipid Res 2005, 46, 1248–1256.

  • 7. Fisher A.B., Dodia C., Manevich Y., Chen J.W., Feinstein S.I.: Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase. J Biol Chem 1999, 274, 21326–21334.

  • 8. Fisher A.B., Dodia C.: Role of phospholipase A2 enzymes in degradation of dipalmitoylphosphatidylcholine by granular pneumocytes. J Lipid Res 1996, 37, 1057–1064.

  • 9. Hernández P., Zomeño L., Ariño B., Blasco A.: Antioxidant, lipolytic and proteolytic enzyme activities in pork meat from different genotypes. Meat Sci 2004, 66, 525–529.

  • 10. Jo M., Yun H.M., Park K.R., Hee P.M., Myoung K.T., Ho P.J., Jae L.S., Moon D.C., Park C.W., Song S., Lee C.K., Bae H.S., Tae H.J.: Lung tumor growth-promoting function of peroxiredoxin 6. Free Radical Biol Med 2013, 61, 453–463.

  • 11. Liu N.N., Liu Z.S., Lu S.Y., Hu P., Li Y.S., Feng X.L., Zhang S.Y., Wang N., Meng Q.F., Yang Y.J., Tang F., Xu Y.M., Zhang W.H., Guo X., Chen X.F., Zhou Y., Ren H.L.: Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries. Vet Immunol Immunopathol 2015, 164, 208–219.

  • 12. Liu N.N., Liu Z.S., Lu S.Y., Hu P., Zhang Ying, Fu B.Q., Li Y.S., Zhou Y., Zhang Yu, Ren H.L.: Isolation and characterisation of peroxiredoxin 6 promoter from sheep (Ovis aries). J Vet Res 2016, 60, 315–321.

  • 13. Manevich Y., Fisher A.B.: Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism. Free Radical Biol Med 2005, 38, 1422–1432.

  • 14. Wu Y.Z., Manevich Y., Baldwin J.L., Dodia C., Yu K., Feinstein S.I., Fisher A.B.: Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity. J Biol Chem 2006, 281, 7515–7525.

  • 15. Zhao C., Zhao J., Wang W., Fan Y., Ma C., Zhang D., Lv Y.: Expression of MLAA34-HSP70 fusion gene constructed by SOE-PCR. Pakistan J Sci 2017, 30, 1125.


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