The characterization, expression and activity analysis of superoxide dismutases (SODs) from Procambarus clarkii

The superoxide dismutases (SODs) are important antioxidant enzymes to remove excess biologically reactive oxygen intermediates, and thus avoid adverse conditions. In this study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, Procambarus clarkii. The open reading frame (ORF) of ecCuZnSOD is 612 bp encoding a 204 amino acid (aa) protein with a 24 aa signal peptide, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein with a 20 aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P. clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. The mRNA expressions of three SODs of P. clarkii (ecCuZnSOD, mtMnSOD and cytosolic MnSOD (cytMnSOD)) were studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulations. After S. eriocheiris challenge, the mRNAs of three SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD and cytosolic MnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNA expressions of three SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila.