实验海洋生物学重点实验室知识库

       补体系统是固有免疫的重要组成部分，在无脊椎动物免疫防御过程中发挥了重要作用。尽管无脊椎动物中已经发现了补体分子类似物，但补体凝集素途径的分子组成和激活机制尚不清楚。本研究采用基因克隆、RT-PCR、免疫组化及原核重组表达等分子生物学技术，对长牡蛎的C型凝集素（CgCLec-2~-4）及甘露糖结合凝集素相关丝氨酸蛋白酶（mannose-binding lectin associated serine protease，MASP）类似分子（CgMASPL-1~-5）的结构和功能进行了研究，初步探讨长牡蛎中可能存在的补体系统凝集素途径。
        根据长牡蛎基因组序列信息，分别克隆获得了CgCLec-2、CgCLec-3和CgCLec-4基因，CgCLec-2和CgCLec-4含有典型的C型凝集素样结构域（C-type lectin-like domain，CTLD），其糖识别基序分别为EPN和EPA，而CgCLec-3缺乏完整的CTLD和典型的糖识别基序。CgCLec-2和CgCLec-3主要在外套膜或者鳃中表达，灿烂弧菌（Vibrio splendidus）和鳗弧菌（Vibrio anguillarum）刺激可诱导这三个分子mRNA表达水平的显著上升。三种CgCLecs重组蛋白均具有广谱的PAMPs识别与结合活性，且rCgCLec-2具有凝集金黄色葡萄球菌（Staphylococcus aureus）的活性，rCgCLec-3和rCgCLec-4对多种革兰氏阴性菌、革兰氏阳性菌和真菌表现出凝集活性。此外，3种rCgCLecs均可以显著促进牡蛎血淋巴细胞对灿烂弧菌的吞噬。rCgCLec-2可显著抑制金黄色葡萄球菌的生长，rCgCLec-3可抑制大肠杆菌（Escherichia coli）和金黄色葡萄球菌（S. aureus）的生长，并对金黄色葡萄球菌（S. aureus）的细胞结构有破坏作用。结果表明，CgCLec-2、CgCLec-3和CgCLec-4不仅作为模式识别受体参与免疫识别，并且可通过调理作用、抑制微生物生长或者破坏细胞结构等途径参与宿主对微生物的清除，在长牡蛎的免疫防御中发挥重要作用。
         从长牡蛎中克隆获得了五个CgMASPLs基因，发现CgMASPL-1~-5均含有丝氨酸蛋白酶结构域（serine protease domain，SPD），其H链各不相同且都缺乏高等动物同源分子保守的CCP结构域，提示低等无脊椎动物MASP分子的结构多样性及低保守性。以偶氮酪蛋白为底物检测它们的丝氨酸蛋白酶活性，发现缺乏典型S位点且以酶原形式参与检测的rCgMASPL-1和酶原形式的rCgMASP-2分子具有较低的丝氨酸蛋白酶活性，而其他分子的SPD结构均具有较高的丝氨酸蛋白酶活性。利用Pull-down技术研究了rCgCLec-2与不同rCgMASPLs分子间的相互作用，发现其可与含有CUB结构域的rCgMASPL-1及rCgMASP-2分子结合，形成类似高等动物collectin/MASP的复合物，提示长牡蛎中可能存在原始的补体系统凝集素途径。
       综上所述，长牡蛎CgCLecs作为长牡蛎固有免疫中重要的模式识别受体参与了对外源微生物的免疫识别和清除。CgCLecs和CgMASPLs在结构和功能上分别与高等动物补体系统中的甘露糖结合凝集素和MASP有一定相似性，且CgCLec-2可与CgMASPLs相互作用，提示长牡蛎中可能存在原始的补体系统凝集素途径。研究结果为进一步探讨长牡蛎补体系统的分子组成及凝集素介导的补体激活机制，揭示无脊椎动物补体系统的进化脉络奠定了基础。

The complement system, as an important part of innate immunity, plays a crucial role in invertebrate immune defense. Although several complement molecule homologs have been found in invertebrates, the consitution and the activation mechanism of the lectin pathway in complement system are still unclear. In the present research, the structures and functions of C-type lectins (CgCLec-2, -3 and -4) and mannose binding lectin associated serine proteases-like (MASPLs) molecules (CgMASPL-1~-5) in Crassostrea gigas was illustrated by gene cloning, RT-PCR, immunohistochemistry and prokaryotic expression etc., aiming at exploring the possible lectin complement pathway in oysters.
The structures of CgCLec-2, CgCLec-3 and CgCLec-4 were analysed based on their cDNA sequences. There was a typical C-type lectin-like domain (CTLD) with carbohydrate recognition motif of EPN and EPA in CgCLec-2 and CgCLec-4 respectively, while CgCLec-3 was lacking of the CTLD and typical carbohydrate recognition motif. By realtime RT-PCR and immunohistochemistry, CgCLec-2 and CgCLec-3 were mainly detected in the mantle or gill, and the mRNA levels of CgCLecs significantly increased after Vibrio splendidus and Vibrio anguillarum challenge. The recombinant CgCLec-2 (rCgCLec-2), CgCLec-3 (rCgCLec-3) and CgCLec-4 (rCgCLec-4) displayed not only broad recognition and binding spectrums to diverse PAMPs and microbes, but also microbial agglutination activities. rCgCLec-2 could agglutinate Staphylococcus aureus, while rCgCLec-3 and rCgCLec-4 exhibited agglutination activities to a variety of Gram-negative bacteria, Gram-positive bacteria and fungi. In addition, they could significantly promote the phagocytosis of heamocytes to V. splendidus. Meanwhile, rCgCLec-2 inhibited the growth of S. aureus, while rCgCLec-3 could inhibit the growth of both Escherichia coli and S. aureus and damage the cell structure of S. aureus. These results indicated that CgCLec-2, CgCLec-3 and CgCLec-4 could not only be involved in immune recognition as pattern recognition receptors (PRRs), but also took part in immune elimination by opsonization, microbial growth suppression or destroying the cell structure of microbes, playing important roles in the host immune response.
The structure analysis of CgMASPLs suggested that the domain orgnization of CgMASPLs were of great diversity. Except for the serine protease domain (SPD) in common, CgMASPLs differed in their H chains and were lack of the CCP domain, which was conserved in the homologous molecules of vertebrates, suggesting that invertebrate MASPs might be characteriaed as higher diversity and lower conservation in sequence structure. Azo-casein was used as the substrate to detect the serine protease activities, and the results showed that the activities of rCgMASPL-1 and rCgMASPL-5 were lower than the other CgMASPLs. There was no typical S in rCgMASPL-1 and both of rCgMASPL-1 and rCgMASPL-5 were zymogens. Furthermore, the interactions between rCgCLec-2 and rCgMASPL-1 or rCgMASPL-2 were confirmed by the assay of Pull-down. rCgCLec-2 and rCgMASPL-1 or rCgMASPL-2 might form a collectin/MASP complex similar to that in higher vertebrates and be involved in the activation of lectin pathway in complement system of oysters.
In summary, CgCLecs were involved in immune recognition and immune elimination. The CgCLecs and CgMASPLs shared structural and functional similarity with vertebrate mannose binding lectins (MBLs) and MASPs, respectively. The interaction of CgCLec-2 and CgMASPLs indicated that the complement lectin pathway existed in C. gigas. These results would contribute to the research on the consitution and activation mechanism of the lectin pawthway in complement system of oysters, and provided clues on the evolutional skeleton of invertebrate complement system.