The Identification Of C-type Lectin CgCLec-TM2 And Preliminary Study On The Immune Function In Crassostrea Gigas

C-type lectins（CTLs）are a superfamily of Ca²⁺-dependent carbohydrate-recognition proteins,which widely participate in nonself-recognition and triggering the signal transduction pathways in the innate immunity.In marine invertebrates,C-type lectins have diverse structures and functions,which play an important role in identifying pathogens,activating immune signaling pathways,and regulating the synthesis of effector molecules.In the present study,using Crassostrea gigas as research object and use bioinformatics,molecular biology and immunology to study the molecular characteristic and immunological function of C-type lectin（Cg CLec-TM2）which containing a single carbohydrate-recognition domain（CRD）and transmembrane domain.The main findings are as follows:A novel CTL（Designated as Cg CLec-TM2）was identified from the C.gigas.The open reading frame（ORF）of Cg CLec-TM2 was of 789 bp encoding a polypeptide of 263 amino acids.The Cg CLec-TM2 was predicted with a molecular mass of 29.91 k Da and a theoretical isoelectric point of 5.16.Cg CLec-TM2 contains a 366 bp CRD,also known as C-type lectin domain（CLECT）.There were seven conserved cysteines in CRD of Cg CLec-TM2.Two novel EFG and FVN motifs were found in Ca²⁺-binding site 2 of Cg CLec-TM2.The m RNA transcripts of Cg CLec-TM2 were detected in hepatopancreas,mantle,gill,haemocytes,adductor muscle and labial palp,and the highest m RNA expression level in haemocytes,which was 94.41-fold（p<0.01）of that in adductor muscle.The relative m RNA expression level of Cg CLec-TM2 in haemocytes significantly up-regulated at 6,24 and 48 h after the stimulation of Vibrio splendidus,which was 4.94-and 12.77-fold of that in control group（p<0.01）,respectively.The recombinant CRD of Cg CLec-TM2（r CRD）was obtained by prokaryotic expression,which able to bind lipopolysaccharide（LPS）,mannose（MAN）,peptidoglycan（PGN）,and poly（I:C）in a Ca²⁺-dependent manner.The r CRD exhibited binding activity to Gram-negative bacteria（V.splendidus,Vibrio anguillarum and Escherichia coli）,Gram-positive bacteria（Staphylococcus aureus,Bacillus subtilis and Micrococcus luteus）and fungi（Pichia pastoris）in a Ca²⁺-dependent manner and exhibited agglutination activity to Gram-negative bacteria（E.coli and V.splendidus）,Gram-positive bacteria（S.aureus and M.luteus）and fungi（P.pastoris）in a Ca²⁺-dependent manner.Besides,the growth of Gram-negative bacteria（E.coli and V.splendidus）were inhibited by r CRD.The phagocytosis rate of haemocytes towards V.splendidus significantly down-regulated from 27.2%to 20.9%after treatment of anti-Cg CLec-TM2-CRD antibody.After the expression of Cg CLec-TM2 was inhibited by RNAi,the expression level of phospho-extracellular regulated protein kinases（p-Cg ERK）in haemocytes,and the m RNA expressions of interleukin17s（Interleukin-17,Cg IL17-1 and Cg IL17-4）decreased significantly after V.splendidus stimulation.The m RNA expressions of Cg IL17-1 and Cg IL17-4 were 0.39-fold（p<0.01）and 0.47-fold（p<0.01）of that in EGFP-RNAi oysters,respectively.These results suggested that Cg CLec-TM2 with novel Ca²⁺-binding motifs served as a pattern recognition receptor（PRR）involved in the recognition of microorganisms,and induce the Cg IL17s expression through regulate the expression of p-Cg ERK in the immune defense.The study enriches the study of the immunological function of C-type lectins in oyster,and of important implications for further study of the immunological mechanisms of marine invertebrates.