| The complement system, as an important part of innate immunity, plays a crucial role in invertebrate immune defense. Although several complement molecule homologs had been found in invertebrates, the consitution and the activation mechanism of the lectin pathway in complement system were 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 to exploring the possible lectin complement pathway in invertebrates.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 PAMPs and microbes, but also microbial agglutination activities. rCgCLec-2 could agglutinate Staphylococcus aureus, while rCgCLec-3 and rCgCLec-4 showed 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, and playing important roles in the host immune response.The structure analysis of CgMASPLs suggested that the domain orgnization of CgMASPLs were of great diversity. Excepted for the serine protease domain(SPD) in common, their H chains were different and the CCP domain was absent, which was appeared in the homologous molecule of vertebrates, suggesting that the MASP structures of invertebrates were more diverse and lower conserved. 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.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. The interaction of CgCLec-2 and CgMASPLs indicated that the lectin pathway in complement existed in C. gigas. These results contributed to the research on the consitution and activation mechanism of the lectin pawthway in complement system of C. gigas, and provided new information on the evolution origin of invertebrate complement system. |
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