| As the important aquaculture species, decapods crustacean can creat great economic value each year. However, the crustacean aquaculture is influenced greatly by the disease. To prevent pathogen invading, the study on crustacean immunity, especially the pathogen recognition mechanism is needed. C-type lectins are expressed in crustaceans with great abundance. However, the functions of them are not clear. To clarify the roles of them in pathogen resisting and to find whether they exert specificity, we studied the accurate functions of several decapods crustacean C-type lectins from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Besides, we also investigated the diversity of the C-type lectin family from kuruma shrimp Marsupenaeus japonicus.1. FcLec3from Chinese white shrimp functions as virus receptor to participate in antiviral responseWe identified a new C-type lectin FcLec3from Chinse white shrimp F. chinensis. The full length of FcLec3cDNA is739bp with the open reading frame of474bp encoding a protein of157amino acids which contains a17aa signal peptide and a C-type lectin like domain. FcLec3is specificly expressed in the fibrillar cells of hepatopancreas. The expression of FcLec3is upregulated by Ⅴ. anguillarum challenge and then goes back to the normal level. Differnently, the expression of FcLec3shows a dual up-regulation post white spot sydrom virus (WSSV) challenge. Recombinant FcLec3can agglutinate bacteria in the presence of calcium, and bind to maltose, N-A-D-mannosamine and maltose, peptidoglycan and muramic acid. Moreover, we find FcLec3can bind to VP28, the most abundant envelope protein of WSSV, suggesting it may function as a virus receptor.2. FcLec4from Chinses white shrimp functions as opsonin to promote phagocytosisWe identified a C-type lectin from Chinese white shrimp F. chinensis, and named it as FcLec4. FcLec4contains a signal peptide and a C-type lectin like domain. Though from crustacean, FcLec4shares high similarity with the lectins from insects. Different from most crustacean C-type lectins, FcLec4is widely distributed in several tissues, including gills, stomach, hepatopancreas and intestine. The expression of FcLec4is induced at both mRNA and protein level post Vibrio anguillarum challenge; besides, the FcLec4protein is serected into circulating system to exert its function. FcLec4can bind to peptidoglycan, agglutinate and tightly bind to several kinds of microorganisms. The binding of FcLec4to Ⅴ. anguillarum can promote the clearance of the bacteria in vivo. We also find a homolog of FcLec4(hFcLec4) which shares94%similarity and presents similar expression profiles with FcLec4in kuruma shrimp M. japonicas. We purified the native protein of FcLec4, and found that it can also bind to Ⅴ. anguillarum and facilitate the clearance in vivo in kuruma shrimp. In addition, this effect is also aiming at several other kinds of bacteria. The native FcLec4can bind to the hemocytes of kuruma shrimp in vivo, suggesting that it may act as an opsonin to promote the hemocytes phagocytosis after binding to the pathogens.3. PcLec2from red swamp crayfish functions as an upstream detector of prophenoloxidase activating systemThrough the sequencing and analysis of the cDNA library from red swamp crayfish P. clarkii, we obtained a C-type lectin designated PcLec2. PcLec2contains a131aa C-type lectin like domain. The expression of PcLec2is restricted in hepatopancreas and greatly induced by lipopolysaccharides and bacteria, suggesting it may be involved in the antibacterial response. Though the recombinant PcLec2can not agglutinate bacteria or inhibit its growth, it can bind to bacteria in dependence of calcium. Besides, calcium can enhance the binding activity. PcLec2can also bind to lipopolysaccharides, however, calcium dose not show the enhancing effect. PcLec2can induce the activation of phenol oxidase activating sysyem both in vitro and in vivo, and also can enhance the induction caused by the bacteria infection in vivo.4. C-type lectins from kuruma shrimp show sequence diversity and pathogen specificityThrough a transcriptome sequence of kuruma shrimp, we identified31putative C-type lectins. By analyzing the sequence information of these lectins, we find that the residues important for maintaining the structure of the CTLD domain, the cystine residues froming the disulfide bonds and the residues involved in the hydrophobic cores for instance, are conserved, while those essential for substrate binding motif vary a lot. This may bring the functional diversity of the C-type lectin family. Most C-type lectins are mainly expressed in hepatopancreas and hemocytes. Those highly expressed in hepatopancreas are more inclined to be tissue specific, while those highly expressed in other tissues are more inclined to be widely distributed. There are21lectins responding to Ⅴ. anguillarum challenge obviously, and most of them respond at the early stage (6h) post infection. The expression of sensitive genes in hepatopancreas is up-regulated post bacterial challenge, while in hemocytes the case is reverse. After WSSV challenge, the expression of13C-type lectin genes were obviously changed. Among them,7lectins respond at the early stage, while the other6genes respond at the mid or late stage (24-48h) post infection.Abundant C-type lectins exist in crustaceans and exert kinds of functions. C-type lectins show not only structural diversity but also pathogen specificity. These findings provide the basis to discuss the significance of the abundance of invertebrate C-type lectins and the "specificity" of innate immunity. |
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