Identification and analysis of icCu/Zn-SOD, Mn-SOD and ecCu/Zn-SOD in superoxide dismutase multigene family of Pseudosciaena crocea

Superoxide dismutases (SODs) belong to a significant and ubiquitous family of metalloenzymes for eliminating excess reactive oxygen species (ROS). In this paper, the complete open reading frames (ORFs) of intracellular Cu/Zn-SOD (icCu/Zn-SOD), Mn-SOD and extracellular Cu/Zn-SOD (ecCu/Zn-SOD) were identified from the large yellow croaker (Pseudosciaena crocea, designated as LycSOD1, LycSOD2 and LycSOD3). The sequences were 465 bp, 678 bp and 645 bp (GenBank accession no. KJ908287, KJ908285 and KJ908286), encoding 154, 225 and 215 amino acid (aa) residues respectively. The deduced aa sequences of LycSOD1, LycSOD2 and LycSOD3 shared high identity to the known icCu/Zn-SODs, Mn-SODs and ecCu/Zn-SODs with BLASTp and Phylogenetic analysis. Two conserved Cu-/Zn-binding sites (H-44, H-47, H-64, H-121 for Cu binding and H-64, H-72, H-81, D-84 for Zn binding in LycSOD1, H-98, H-100, H-115, H-164 for Cu binding and H-115, H-163, H-166, D-169 for Zn binding in LycSOD3) and one conserved manganese coordinating sites (H-57, H-101, D-186, H-190 in LycSOD2) were identified. The total length of DNA sequences of LycSOD1, LycSOD2 and LycSOD3 were 3447 bp, 3387 bp and 3886 bp respectively, and there were 4 introns and 5 exons in Cu/Zn-SODs (LycSOD1 and LycSOD3), but only 3 exons and 2 introns in LycSOD3. Spatial expression analysis indicated the highest mRNA expression of three SODs all appeared in liver among eight detected tissues, the highest expression level was LycSOD1, then LycSOD2 and the lowest was LycSOD3 for almost each tissue. The expression of LycSOD1, LycSOD2 and LycSOD3 mRNA were all up-regulated in liver after Vibrio alginolyticus stimulation. The temporal expression peak of LycSOD1 and LycSOD2 were around 9-fold and 8-fold compared to control respectively, whereas, LycSOD3 got the highest level at 48 h post-injection (about 4.2-fold). All the results gave several new and useful evidences for further understanding the regulatory mechanism of superoxide dismutases in the innate immune system of sciaenidae fish.