Lv Zhao, Qiu Limei, Liu Zhaoqun, Wang Weilin, Chen Hao, Jia Yunke, Jia Zhihao, Jiang Shuai, Wang Lingling, Song Linsheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China.
Dev Comp Immunol. 2018 Dec;89:152-162. doi: 10.1016/j.dci.2018.08.014. Epub 2018 Aug 22.
Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln, His and Asn). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.
组织蛋白酶L1（CTSL1）是一种具有木瓜蛋白酶样结构的溶酶体半胱氨酸蛋白酶。基于其蛋白水解活性，已知它参与针对病原体感染的免疫反应的多个过程。在本研究中，从太平洋牡蛎中鉴定出一种CTSL1同源物（命名为CgCTSL1）。它包含一个典型的单一Pept_C1结构域，带有三个保守的催化必需残基（谷氨酰胺、组氨酸和天冬酰胺）。CgCTSL1的mRNA在牡蛎组织中普遍表达，在鳃和血细胞等重要免疫组织中的表达水平最高。通过免疫组织化学在鳃和肝胰腺中主要检测到CgCTSL1蛋白。重组CgCTSL1（rCgCTSL1）表现出蛋白水解活性，以剂量依赖性方式切割底物Ac-FR-氨基-4-三氟甲基香豆素（AFC），并且抑制剂可以降低其蛋白水解活性。在干扰CgCTSL1 mRNA后，牡蛎血细胞的蛋白水解活性显著下调，释放的AFC荧光值从375.84降至179.21（p < 0.05）。流式细胞术分析显示，与非吞噬性血细胞的平均荧光强度（MFI）值5,130相比，吞噬细胞中CgCTSL1蛋白的表达更高，MFI值为21,187（4.13倍，p < 0.01）。进一步的共聚焦分析表明，带有绿色珠子信号的活跃吞噬血细胞与更强的CgCTSL1阳性信号共定位。粒细胞和半粒细胞样吞噬细胞亚群中CgCTSL1的mRNA表达水平分别是无粒细胞中的298.12倍（p < 0.01）和2.75倍（p < 0.01）。血细胞蛋白的蛋白质印迹分析表明，与无粒细胞相比，CgCTSL1在粒细胞和半粒细胞中相对丰富。这些结果共同表明，CgCTSL1是一种在吞噬细胞样血细胞中高表达的CTSL1同源物，可能因其保守的蛋白水解活性参与细胞免疫反应，这可能为吞噬细胞与非吞噬性血细胞之间的差异以及太平洋牡蛎中吞噬细胞有前景的分子标记物的鉴定提供线索。
