State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, PR China; Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, PR China.
Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science, Hainan University, PR China.
Fish Shellfish Immunol. 2022 Sep;128:316-326. doi: 10.1016/j.fsi.2022.07.078. Epub 2022 Aug 8.
Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys, His, and Asn). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn and Ca but inhibited by Fe and Cu. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1β), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.
组织蛋白酶作为一类蛋白水解酶，广泛存在于许多组织的溶酶体中，在各种生理活动中发挥着重要作用。组织蛋白酶 C（CTSC）是一种溶酶体半胱氨酸蛋白酶，是溶酶体水解酶家族的重要组成部分。本研究从卵形鲳鲹（Trachinotus ovatus）中鉴定出 CTSC（TroCTSC），并分析了其功能。TroCTSC 含有一个 1368bp 的开放阅读框，编码 455 个氨基酸，其中包含三个保守的催化活性位点（Cys、His 和 Asn）。它与其他已知的硬骨鱼类 CTSC 序列具有高度同源性（69.47%-90.77%），与 Seriola dumerili 最为密切相关。TroCTSC 在肌肉、肝脏和头肾中表达量最高。在哈维弧菌感染后，肝脏、脾脏和头肾中 TroCTSC 的表达水平显著上调。在金鲳鱼吻突细胞（GPS）中发现 TroCTSC 存在于细胞质中，其中一些与溶酶体共定位。在受到哈维弧菌刺激后，TroCTSC 被转运到细胞核中。体外实验结果表明，重组蛋白 rTroCTSC 的最佳水解酶活性在 40°C 和 pH 5.5 时达到最大。rTroCTSC 的活性受到 Zn 和 Ca 的促进，但受到 Fe 和 Cu 的抑制。然而，三个突变蛋白 rTroCTSC-C251A、rTroCTSC-H397A 和 rTroCTSC-N419A 的酶活性显著降低。此外，体内实验结果表明，过表达 TroCTSC 可显著增强机体抵抗哈维弧菌的能力，并促进促炎细胞因子白细胞介素 1-β（IL-1β）、白细胞介素 6（IL-6）、白细胞介素 8（IL-8）、干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）的表达。相反，干扰 TroCTSC 的表达会导致哈维弧菌感染后细菌数量显著增加。我们的研究结果表明，TroCTSC 是先天免疫系统的重要效应因子，在抗菌免疫中发挥关键作用。
