The molecular characterizations of Cu/ZnSOD and MnSOD and its responses of mRNA expression and enzyme activity to Aeromonas hydrophila or lipopolysaccharide challenge in Qihe crucian carp Carassius auratus

Abstract

Superoxide dismutases (SODs), as the prime antioxidant enzymes, present the first line of defense against oxidative stress caused by excessive reactive oxygen species (ROS) in organism. In the study, two distinct members of SOD family were cloned and analyzed in Qihe crucian carp Carassius auratus (designated as CaCu/ZnSOD and CaMnSOD, respectively). The full-length cDNA of CaCu/ZnSOD is 759 bp, containing a 5' -untranslated region (UTR) of 39 bp, a ORF (including stop codon, TAG) of 465 bp and a 3'-UTR of 255 bp. The ORF of CaCu/ZnSOD encodes a protein of 154 amino acids (aa), in which, two Cu/ZnSOD signature (⁴⁵GFHVHAFGDNT⁵⁵ and ¹³⁹GNAGGRLACGVI¹⁵⁰) and four conserved amino acids for Cu/Zn²⁺-binding sites (H64, H72, H81 and D84) were observed. The full-length CaMnSOD cDNA (960 bp) consists of a 5'-UTR of 114 bp, a ORF of 675 bp and a 3'-UTR of 231 bp, the ORF of CaMnSOD encodes a 224 aa protein with a 26 aa mitochondrial-targeting sequence (MTS) in the N-terminus, and four conserved amino acids for manganese binding (H52, H100, D185 and H189) were observed. Multiple alignment and the structural analysis revealed two Cu/ZnSOD signature motifs and a MnSOD signature motif as well as the invariant binding sites for Cu²⁺/Zn²⁺ in CaCu/ZnSOD and Mn²⁺ in CaMnSOD. The phylogenetic analysis indicated that CaCu/ZnSOD was homologous to cytosolic Cu/ZnSODs, and CaMnSOD was high similarity with mitochondrial MnSODs from other fish. The tissue distribution analysis demonstrated that CaCu/ZnSOD and CaMnSOD were highly expressed in liver, heart and muscle. The dynamic expressions of CaCu/ZnSOD and CaMnSOD were observed after the challenges with Aeromonas hydrophila or LPS, which generally increased in liver, gill, kidney and spleen, while, the mRNA expressions were down-regulated at some time points in head kidney. The enzyme activities increased after A. hydrophila or LPS challenge, compared to the control. In this study, the molecular structures and functional motifs of CaCu/ZnSOD and CaMnSOD were determined, and it is crucial for us to understand the biological functions of SODs. The highest level in liver showed that the function of liver to remove ROS is much more important. The obvious responses of mRNA expression levels and enzyme activities to pathogens indicate the important roles of CaCu/ZnSOD and CaMnSOD in antioxidant defense in C. auratus.

Keywords: Aeromonas hydrophila; Carassius auratus; Lipopolysaccharide; Superoxide dismutase.