State Key Laboratory of Marine Environmental Science, College of Ocean & Earth Sciences, Xiamen University, Xiamen, Fujian, China.
State Key Laboratory of Marine Environmental Science, College of Ocean & Earth Sciences, Xiamen University, Xiamen, Fujian, China; State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen, Fujian, China; Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen University, Xiamen, Fujian, China.
Fish Shellfish Immunol. 2020 Aug;103:442-453. doi: 10.1016/j.fsi.2020.05.045. Epub 2020 May 21.
Apoptosis plays a key role in the immune defense against pathogen infection, and caspase is one of the most important protease enzyme families, which could initiate and execute apoptosis. Among crustaceans, several caspase genes have been reported. However, caspase in mud crab Scylla paramamosain, have not been identified yet. Here, in the present study, we characterized a new caspase, named as Sp-caspase, from S. paramamosain. The full-length cDNA sequence of Sp-caspase contained 966 bp open reading frame, encoding 322 amino acids, and its molecular weight was 36 kDa. This gene has three conserved domains of the caspase family, a prodomain, a large subunit P20 and a small subunit P10. Phylogenetic analysis showed that Sp-caspase was clustered into an effector caspase group. Sp-caspase mainly distributed in midgut, hepatopancreas, hemocytes and female ovaries, and the transcript was significantly regulated in different tissues after being challenged with Vibrio parahaemolyticus, Vibrio alginolyticus or LPS. After infection with V. alginolyticus, the apoptosis rate of hemocytes notably increased, while the mRNA level of Sp-caspase and hydrolysis activity of caspase 3/7 significantly decreased. Furthermore, in vitro assays showed that the recombinant protein tSp-caspase (deletion of Sp-caspase prodomain) could efficiently recognize and cleave human caspase 3/7 substrate Ac-DEVD-pNA, functioning as an effector caspase. Meanwhile, heterologous expression of Sp-caspase in several cell lines (HEK293T cells, HeLa cells and HighFive cells) could specifically induce cell apoptosis. Taken together, these data demonstrated that Sp-caspase could perform apoptosis as an effector caspase. In addition, it might be a negative regulator of hemocytes apoptosis under pathogen infection, which would contribute to homeostasis and immune defense of hemocytes in S. paramamosain.
细胞凋亡在宿主抵御病原体感染的免疫防御中起着关键作用，而半胱天冬酶是最重要的蛋白酶家族之一，能够启动和执行细胞凋亡。在甲壳动物中，已经报道了几种半胱天冬酶基因。然而，锯缘青蟹 Scylla paramamosain 中的半胱天冬酶尚未被鉴定。在本研究中，我们从锯缘青蟹中鉴定了一种新的半胱天冬酶，命名为 Sp-caspase。Sp-caspase 的全长 cDNA 序列包含 966 bp 的开放阅读框，编码 322 个氨基酸，其分子量为 36 kDa。该基因具有半胱天冬酶家族的三个保守结构域，即前导肽、大亚基 P20 和小亚基 P10。系统进化分析表明，Sp-caspase 聚类为效应子半胱天冬酶组。Sp-caspase 主要分布于中肠、肝胰腺、血细胞和雌性卵巢，且在受到副溶血弧菌、溶藻弧菌或 LPS 刺激后，在不同组织中转录水平显著调节。感染溶藻弧菌后，血细胞的凋亡率显著增加，而 Sp-caspase 的 mRNA 水平和 caspase 3/7 的水解活性显著降低。此外，体外实验表明，重组蛋白 tSp-caspase（缺失 Sp-caspase 前导肽）能够有效地识别并切割人 caspase 3/7 底物 Ac-DEVD-pNA，作为效应子半胱天冬酶发挥作用。同时，Sp-caspase 在几种细胞系（HEK293T 细胞、HeLa 细胞和 HighFive 细胞）中的异源表达能够特异性诱导细胞凋亡。综上所述，这些数据表明 Sp-caspase 可以作为效应子半胱天冬酶执行细胞凋亡。此外，它可能是病原体感染下血细胞凋亡的负调控因子，有助于维持锯缘青蟹血细胞的内稳态和免疫防御。
