The Study Of Immune Function And Molecular Recognition Mechanism Of Bivalve C-type Lectins

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that consist at least one carbohydrate-recognition domain(CRD). C-typle lectins are one of the most important pattern-recognition receptors(PRR) in innate immunity. In this thesis, by using molecular biology, immunology and bioinformatics methods, several structurally different bivalve C-type lectins including multi-CRD containing C-type lectin(Cf Lec-4), single-CRD containing C-type lectin(AiCTL-2, AiCTL-3, AiCTL-4 and AiCTL-7) and C-type lectin anchoring on the membrane(CgCLec-5), were cloned from Chlamys farreri, Argopectens irradians and Crassostrea gigas and recombinant expressed. Their immune functions, as well as recognition mechanism, were comprehensively explored.CfLec-4 was the only lectin found in C. farreri that contains four CRDs with EPD/LSD, EPN/FAD, EPN/LND and EPN/YND key binding motifs. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated after the stimulations of β-glucan, LPS or PGN. CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4(designated as rCflec-4) could bind to LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broad microbe binding spectrum towards Gram-positive bacteria S. aureus and M. luteu, Gram-negative bacteria E. coli, V. anguillarum, and fungi P. pastoris. Meanwhile, rCf Lec-4 could significantly enhance the phagocytosis of C. farreri hemocytes in vitro.The recombiant proteins of four CRDs(designated as rCRD1, rCRD2, rCRD3 and rCRD4) had different PAMPs binding spectrum and affinity. rCRD3 and rCRD4 could bind to all four PAMPs tested while rCRD1 and rCRD2 could only bind to LPS and mannan. Comparing their affinity towards single PAMP, it could be found that they display different binding affinity. rCRD3 has the strongest binding affinity towards LPS, glucan, and mannan while rCRD4 had the strongest binding affinity towards PGN. Similarly, the microbe binding spectrum of four CRDs was also different, in which rCRD3 had the same microbe binding spectrum as rCf Lec-4. Furthermore, rCRD1, rCRD3 and rCRD4 could significantly enhance the phagocytosis of hemocytes against E. coli. These results collectively showed that the four CRDs of Cf Lec-4 had differentiation in immune function, reflecting both in the PAMPs or microbes binding affinity and in phagocytosis enhancement activity.The immune function of the A. irradians C-type lectin AiCTL-7 which contains EPD/WSD motif was investigated. The recombinant protein of AiCTL-7(designated as rAiCTL-7) could bind to LPS, PGN, mannan, yeast glucan and poly I:C in vitro, and displayed a broad microbes binding spectrum towards Gram-positive bacteria S. aureus, Gram-negative bacteria E. coli, V. anguillarum as well as fungi P. pastoris and Y. lipolytica. The monosaccharide binding affinity test showed that rAiCTL-7 could bind both D-mannose and D-galactose. Meanwhile, it could agglutinate S. aureus, V. anguillarum, Y. lipolytica and rabbit erythrocyte. rAiCYL-7 could also inhibit the growth of E. coli in vitro. As a pattern-recognition receptor, the EPD/WSD motif endowed AiCTL-7 broad PAMPs binding spectrum and microbes binding spectrum.Three C-type lectins with EPN key motif in Ca2+ binding site 2 were recombinant expressed and their immune functions were studied. The mRNA transcripts of the three AiCTLs could be detected in all tested tissues including hepatopancreas, gonad, adductor muscle, heart, hemocytes, mantle, and gill, with the highest expression level in hepatopancreas. The recombinant protein of AiCTL-2, AiCTL-3 and AiCTL-4(designated as rAiCTL-2, rAiCTL-3 and rAiCTL-4) had similar PAMPs binding pattern. rAiCTL-3 and rAiCTL-4 could bind LPS, PGN, and glucan in vitro, while rAiCTL-2 could bind LPS and PGN. None of them had binding affinity towards mannan. However, each of them showed different binding affinity towards the same PAMP. rAiCTL-2 had strongest binding affinity towards LPS and PGN while rAiCTL-4 had strongest binding affinity towards glucan. And they could bind microbes with different binding spectrum in which rAiCTL-3 had broader microbe binding spectrum than r AiCTL-2 and rAiCT-4. rAiCTL-3 could bind S. aureus, E. coli and V. anguillarum while rAiCTL-2 and rAiCTL-4 could only bind S. aureus. All three C-type lectins could significantly enhance the phagocytosis of A. irradians hemocytes against E. coli in vitro.CgCLec-5 was mainly located in the ganglion, gill and mantle of C. gigas. It was also found on the cell surface of hemocytes. The CRD of CgCLec-5 was recombinant expressed to investigate its recognition pattern. The recombinant expressed CRD of CgCLec-5 could bind LPS, PGN and mannan. CgCLec-5 was found significantly enhanced the phagocytosis of C. gigas hemocytes.C-type lectins studied in this thesis could function as pattern-recognition receptor to recognize and bind certain PAMPs and microbes. Some of them could agglutinate microbes and blood cells. Moreover, C-type lectin could function as opsonin. These results proved that C-type lectins played an important role in bivalve innate immunity and could help further understand the innate immune mechanism of Bivalvia which could build foundation for illuminating the immune defense mechanism of Mollusca.