CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, 266071, China.
Fish Shellfish Immunol. 2019 Jun;89:98-107. doi: 10.1016/j.fsi.2018.12.047. Epub 2019 Mar 6.
Clip domain serine proteases (cSPs), a family of multifunctional proteins, play a crucial role in innate immune system. Here, we report the functional characterization of two clip domain serine proteases (PtcSP1 and PtcSP3) from the swimming crab Portunus trituberculatus. The recombinant N-terminal clip domains and the C-terminal SP-like domains of PtcSP1 and PtcSP3 were expressed in Escherichia coli system, and assayed for various biological functions: protease activity, antimicrobial activity, bacterial clearance and microbial-binding activity. The recombinant SP-like domains of PtcSP1 and PtcSP3 exhibited trypsin-like protease activity, while their recombinant clip domains showed strong antibacterial activity and could bind to bacteria and yeast, suggesting the potential roles of PtcSP1 and PtcSP3 in immune defense and pattern recognition. Unlike PtcSP3, PtcSP1 revealed the opsonic activity as shown by a higher bacterial clearance rate of Vibrio alginolyticus coated with the combination of the recombinant clip domain and SP-like domain of PtcSP1 as compared with V. alginolyticus only. Knockdown of PtcSP1 or PtcSP3 by RNA interference resulted in a significant decrease of total phenoloxidase (PO) activity in crab, suggesting that PtcSP1 and PtcSP3 are involved in the proPO system. In addition, suppression of PtcSP1 or PtcSP3 changed the expression of PtALFs and complement-like components. All these findings suggest that PtcSP1 and PtcSP3 are multifunctional immune molecules and perform different protective functions in crab defense.
剪接体丝氨酸蛋白酶（cSPs）是一类多功能蛋白，在先天免疫系统中发挥着关键作用。本研究报道了来自三疣梭子蟹（Portunus trituberculatus）的两种剪接体丝氨酸蛋白酶（PtcSP1 和 PtcSP3）的功能特征。PtcSP1 和 PtcSP3 的重组 N 端剪接体结构域和 C 端 SP 样结构域在大肠杆菌系统中表达，并进行了各种生物学功能的测定：蛋白酶活性、抗菌活性、细菌清除率和微生物结合活性。PtcSP1 和 PtcSP3 的重组 SP 样结构域具有胰蛋白酶样蛋白酶活性，而其重组剪接体结构域表现出强烈的抗菌活性，并能与细菌和酵母结合，表明 PtcSP1 和 PtcSP3 在免疫防御和模式识别中具有潜在作用。与 PtcSP3 不同，PtcSP1 表现出调理活性，与仅用 V. alginolyticus 相比，用 PtcSP1 的重组剪接体结构域和 SP 样结构域包被的 V. alginolyticus 的细菌清除率更高。通过 RNA 干扰敲低 PtcSP1 或 PtcSP3 导致蟹总酚氧化酶（PO）活性显著降低，表明 PtcSP1 和 PtcSP3 参与了 proPO 系统。此外，抑制 PtcSP1 或 PtcSP3 改变了 PtALFs 和补体样成分的表达。所有这些发现表明，PtcSP1 和 PtcSP3 是多功能免疫分子，在蟹防御中发挥不同的保护功能。
