College of Fishery, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Zhanjiang, China; Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, China.
College of Fishery, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Zhanjiang, China.
Fish Shellfish Immunol. 2024 Jun;149:109594. doi: 10.1016/j.fsi.2024.109594. Epub 2024 Apr 30.
Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.
非特异性细胞毒性细胞（NCC）是参与硬骨鱼非特异性免疫的重要免疫细胞。作为 NCC 表面的受体分子，非特异性细胞毒性细胞受体蛋白 1（NCCRP-1）被认为在调节其活性方面起着至关重要的作用。然而，关于通过 NCCRP-1 传递信号的信号分子的研究有限。在这项研究中，构建了罗非鱼肝脏和头部肾脏的酵母双杂交（Y2H）文库，并用诱饵载体尼罗罗非鱼的 NCCRP-1（On-NCCRP-1）筛选，以获得与互作蛋白序列的 C 型凝集素（On-CTL）。随后，克隆并分析了 On-CTL 的全长序列。表达分析表明，On-CTL 在肝脏中高度表达，并广泛分布于其他组织中。此外，在受到无乳链球菌攻击后，On-CTL 在大脑、肠和头部肾脏中的表达显著上调。还使用点对点 Y2H 方法来确认 On-NCCRP-1 和 On-CTL 之间的结合。纯化了重组 On-CTL（rOn-CTL）蛋白。体外实验表明，rOn-CTL 通过与 On-NCCRP-1 的相互作用可以上调 NCC 中杀伤效应分子的表达。此外，rOn-CTL 激活 NCC 导致其消除翻车鱼细胞的能力显著增强，表明 rOn-CTL 通过 NCC 受体分子 On-NCCRP-1 有效调节 NCC 的杀伤活性。这些发现极大地促进了我们对 NCC 活性调控机制的理解，为该领域的未来研究铺平了道路。
