Preliminary Study On C-type Lectin Of Sea Cucumber Apostichopus Japonicus And The Mechanism Of Immune Response Mediated By It

C-type lectins（CTLs）are Ca²⁺dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains（CRDs）,which is an important pattern recognition receptor（PRR）in the innate immune system.Using Apostichopus japonicus as material and comprehensive use of bioinformatics,molecular biology and immunology to explore the immune function of C-type lectin containing a single CRD（SJL-1）and C-type lectin containing multiple functional domains（AjCTL-2）in this paper.We discussed the mechanism of sugar recognition that combined with the structural characteristics of C-type lectin,the mechanism of sugar recognition was discussed,and then explained the role of C-type lectin in immune defense of Apostichopus japonicus.The immunological activity of SJL-1 containing DPN/WVD motifs was studied in this paper.We detected that SJL-1 was specific distributed in the tube-foot,body wall and respiratory trees of Apostichopus japonicus.The mRNA expression level of SJL-1 in coelomocyte was up-regulated significantly at 12 h after Vibrio splendidus challenge.The recombinant SJL-1（rSJL-1）displayed binding activity to all examined pathogen-associated molecular patterns（PAMPs）dependent of Ca²⁺,including lipopolysaccharide（LPS）,peptidoglycan（PGN）,mannose（Man）and D-galactose.Moreover,rSJL-1 displayed strong binding abilities to V.splendidus and week binding ability to Vibrio anguillarum,Staphylococcus aureus,Bifidobacterium breve,Pichia pastoris and Yarrowia lipolytica.The results show that SJL-1 could act as a pattern recognition receptor and contain a single CRD.In order to further understand the relationship between the structure and functional characteristics of C-type lectin of Apostichopus japonicus.The immunological function of AjCTL-2 with three different functional domains was studied in this paper.AjCTL-2 contained a canonical signal peptide at the N-terminus,a low density lipoprotein receptor class A（LDLa）,a C1r/C1s/Uegf/bone morphogenetic protein 1（CUB）,and a CRD with two motif Glu-Pro-Asn（EPN）and Trp-Asn-Asp（WND）in Ca²⁺binding site 2.The mRNA transcripts of AjCTL-2 were highly expressed in coelomocyte.The mRNA expression level of AjCTL-2 in coelomocyte increased significantly after Vibrio splendidus stimulation,and dramatically pecked at 12 h.AjCTL-2 protein was mainly detected in cytoplasm of coelomocyte by immunofluorescence.The recombinant AjCTL-2（rAjCTL-2）displayed binding activity to D-galactose independent of Ca²⁺,while binding activity towards other tested pathogen-associated molecular patterns（PAMPs）including LPS,PGN,and Man has not been detected.Surface plasmon resonance（SPR）analysis further revealed the high binding specificity and moderate binding affinity of r AjCTL-2 towards D-galactose(KD=4.093×10^-6).After rAjCTL-2 was blocked by the polyclonal antibody,its binding activity towards D-galactose could not be detected by using a blocking ELISA（B-ELISA）.Moreover,rAjCTL-2 could bind various microorganisms including V.splendidus,Vibrio anguillarum,Staphylococcus aureus,Bifidobacterium breve and Yarrowia lipolytica.These results collectively suggested that C-type lectin of Apostichopus japonicus.could act as PRR that involved in immune defense.This paper laid a foundation for further understanding the structure and function of immune recognition molecules in echinoderms and elucidating the immune defense mechanism of echinoderms.