Liu Huihui, He Jianyu, Chi Changfeng, Gu Yifeng
National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan, 316022, PR China.
National Engineering Research Center of Marine Facilities Aquaculture, Zhejiang Ocean University, Zhoushan, 316022, PR China.
Fish Shellfish Immunol. 2015 Apr;43(2):491-501. doi: 10.1016/j.fsi.2015.01.032. Epub 2015 Jan 31.
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.
超氧化物歧化酶（SODs）属于一个重要且广泛存在的金属酶家族，用于清除过量的活性氧（ROS）。在本文中，从大黄鱼（Pseudosciaena crocea，命名为LycSOD1、LycSOD2和LycSOD3）中鉴定出细胞内铜/锌超氧化物歧化酶（icCu/Zn - SOD）、锰超氧化物歧化酶（Mn - SOD）和细胞外铜/锌超氧化物歧化酶（ecCu/Zn - SOD）的完整开放阅读框（ORFs）。序列长度分别为465 bp、678 bp和645 bp（GenBank登录号：KJ908287、KJ908285和KJ908286），分别编码154、225和215个氨基酸（aa）残基。通过BLASTp和系统发育分析，LycSOD1、LycSOD2和LycSOD3推导的氨基酸序列与已知的icCu/Zn - SODs、Mn - SODs和ecCu/Zn - SODs具有高度同源性。鉴定出两个保守的铜/锌结合位点（LycSOD1中用于结合铜的H - 44、H - 47、H - 64、H - 121以及用于结合锌的H - 64、H - 72、H - 81、D - 84；LycSOD3中用于结合铜的H - 98、H - 100、H - 115、H - 164以及用于结合锌的H - 115、H - 163、H - 166、D - 169）和一个保守的锰配位位点（LycSOD2中的H - 57、H - 101、D - 186、H - 190）。LycSOD1、LycSOD2和LycSOD3的DNA序列总长度分别为3447 bp、3387 bp和3886 bp，铜/锌超氧化物歧化酶（LycSOD1和LycSOD3）中有4个内含子和5个外显子，但LycSOD3中只有3个外显子和2个内含子。空间表达分析表明，在检测的八个组织中，三种超氧化物歧化酶的mRNA表达在肝脏中均最高，几乎在每个组织中，表达水平最高的是LycSOD1，其次是LycSOD2，最低的是LycSOD3。溶藻弧菌刺激后，肝脏中LycSOD1、LycSOD2和LycSOD3 mRNA的表达均上调。LycSOD1和LycSOD2的时间表达峰值分别比对照高约9倍和8倍，而LycSOD3在注射后48小时达到最高水平（约4.2倍）。所有这些结果为进一步了解石首鱼科鱼类先天免疫系统中超氧化物歧化酶的调节机制提供了一些新的有用证据。
