Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China.
Fish Shellfish Immunol. 2014 Jan;36(1):194-205. doi: 10.1016/j.fsi.2013.11.006. Epub 2013 Nov 13.
The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.
木瓜蛋白酶家族的溶酶体半胱氨酸蛋白酶 cathepsin B 是一种关键的调节剂和信号分子，参与多种生物学过程，如细胞凋亡的调节和病毒的激活。在本研究中，从尖吻鲈（Epinephelus coioides）中克隆和表征了 cathepsin B 基因（Ec-CB）。Ec-CB 的全长 cDNA 由 1918bp 组成，编码 330 个氨基酸的多肽，与鱼类和哺乳动物的 cathepsin B 具有较高的同一性。Ec-CB 具有典型的 cathepsin B 结构特征，包括 N 端信号肽、前肽区和半胱氨酸蛋白酶结构域，这些结构域在其他 cathepsin B 序列中保守。系统进化分析表明，Ec-CB 与 Lutjanus argentimaculatus 最为密切相关。RT-PCR 分析显示，Ec-CB 转录本在所有检测的组织中均有表达，在脾脏、肾脏和鳃中表达丰富。在受到新加坡石斑鱼虹彩病毒（SGIV）刺激后，Ec-CB 在尖吻鲈中的 mRNA 表达在感染后 24 小时上调。亚细胞定位分析表明，Ec-CB 主要分布在细胞质中。当鱼细胞（GS 或 FHM）用 cathepsin B 特异性抑制剂 CA-074Me 处理时，SGIV 诱导的 CPE 发生延迟，病毒基因转录明显受到抑制。此外，CA-074Me 还抑制了 FHM 细胞中 SGIV 诱导的典型凋亡。综上所述，我们的结果表明，Ec-CB 在 SGIV 感染中可能发挥功能作用。
