細胞自溶酵素 L (cathepsin L) 在免疫上的角色： 從魚類以及節肢動物的角度進行探討

Abstract

細胞自溶酵素 L (cathepsin L) 是一種存在於溶小體中的蛋白水解酶，參與真核生物中多種重要的生理與病理功能，並在免疫反應中扮演重要角色。同時 cathepsin L 也是一個廣泛存在於動物體內的酵素，從原始的海綿動物到高等哺乳動物皆有。在動物演化中，免疫系統經歷由簡單到複雜的進化過程，cathepsin L 在 這些不同演化程度免疫系統上所扮演的角色。本研究擬探討在不同免疫演化層級 的物種中，cathepsin L 所扮演角色。研究中挑選演化上具有完整先天性與後天性性 免疫反應的的硬骨魚 (點帶石斑魚)，以及兩種僅具先天性免疫反應的節肢動物 (昆蟲綱的黑水虻和軟甲綱的白蝦) 作為模式動物進行研究，闡明不同物種中 cathepsin L 在免疫反應中可能扮演的角色。在點帶石斑魚為研究對象的研究中，透 過免疫組織染色中 (IHC staining) 觀察到 cathepsin L 存在於石斑魚免疫器官中的免疫相關細胞，特別是肝臟中的 Kupffer’s cells 與頭腎中的 leukocyte-like cells。同 時，首次以活體 (in vivo) 實驗證實，當石斑魚體內具有酵素活性之 cathepsin L 增加時，免疫相關的基因受到調控。在黑水虻研究中發現 cathepsin L 基因表現量在 不同蟲齡之間差異，特別在第五齡時，cathepsin L 基因表現最為顯著。IHC staining 證實 cathepsin L 在黑水虻幼蟲體內，存在於擔負消化與免疫功能的中腸中，尤其中中腸最甚。此外，黑水虻幼蟲在受到免疫刺激後，體內的 cathepsin L 無論是在基因表現，還是酵素活性上，皆有顯著上升的趨勢。在白蝦的研究中發現白蝦受到微孢子蟲感染和副溶血弧菌感染，白蝦肝胰腺中的 cathepsin L 基因表現量與肝胰腺組織損傷程度密切相關。綜合以上的研究成果，我們發現 cathepsin L 在此三種 物種中，基因表現量與酵素活性在受到免疫刺激時，皆有上升趨勢，顯示 cathepsin L 在免疫反應中受到調控。此外，在不同演化層級不同的物種，cathepsin L 表現位置有所差異，在演化較為初級的黑水虻與白蝦中，cathepsin L 存在於負責消化與免疫的器官中，暗示其兼具消化與防禦的功能。隨著演化的推進，在較為高等的石斑魚中發現，cathepsin L 存在於免疫器官中，除了降解入侵的病原以外，並透過修飾免疫分子進行免疫調節。

Cathepsin L is a lysosomal protease in eukaryotes, involved in various physiological and pathological functions, and plays a crucial role in immune responses. This hydrolytic protein can be found in both intracellular and extracellular matrices, and its evolution can be traced from sponges to mammals. During animal evolution, the immune system evolved from simple to complex, raising curiosity about the role of cathepsin L in these different evolutionary stages of the immune system. This study aims to investigate the role of cathepsin L in species at different levels of immune evolution. We selected the orange-spotted grouper (the teleost fish Epinephelus coioides), which has a complete innate and adaptive immune response, and two arthropods, including black soldier fly (the insect Hermetia illucens) and the white shrimp (the crustacean Litopenaeus vannamei), which relying solely on innate immunity. We analyzed cathepsin L gene expression, tissue distribution, and enzymatic activity to elucidate the possible roles of cathepsin L in immune responses across different species. In the study of the grouper, IHC staining showed the presence of cathepsin L in immune-related cells in immune organs, particularly in Kupffer’s cells in the liver and leukocyte-like cells in the head kidney. Furthermore, in vivo experiments demonstrated that increased of cathepsin L with enzymatic activity would regulate immune-related gene expression. In the second study of black soldier fly, cathepsin L mRNA levels were highest at the fifth larval stage, compared to those at the second through sixth larval stages. Cathepsin L was localized in the midgut, especially in the middle midgut, responsible for digestion and immune functions. Additionally, the expression level of mRNA and enzymatic activity of cathepsin L significantly increased upon immune stimulation. In the third study of shrimp, cathepsin L gene expression in the hepatopancreas correlated with tissue damage caused by Enterocytozoon hepatopenaei and Vibrio parahaemolyticus infections. Summarizing the findings, we observed that cathepsin L gene expression and enzyme activity increased upon immune stimulation in all three species, suggesting that cathepsin L did regulate immune responses. However, the localization of cathepsin L differed among species. In the more primitive black soldier fly and shrimp, cathepsin L was present in organs responsible for both digestion and immunity, suggesting dual functions. In the more advanced species such as grouper, cathepsin L was found in immune organs, participating in pathogen degradation and the regulation of immune-related genes.