The Study Of Immune Functions And Nonself Recognition Mechanism Of C-type Lectin PtCTL-9 In Portunus Trituberculatus

C-type lectins,as pattern recognition receptor,have an important role in immune system.Carbohydrate-recognition domain（CRD）is Ca²⁺-dependent,which endows C-type lectins with the function of recognizing and scavenging non-self factors.In the present study,a new C-type lectin was identified from Portunus trituberculatus（designated as PtCTL-9 according to the order of discovery）through transcriptome sequencing and bioinformatics methods.This research carried out immune function study comprehensively on PtCTL-9.Mutanting the key motifs and using prokaryotic expression system obtaining target protein and mutant protein to explore the molecular recognition mechanism of PtCTL-9.PtCTL-9 contains a CRD domain according to the multiple sequence comparison.QPN（Gln-Pro-Asn）and FHS（Phe-His-Ser）were identified as the key motifs that determine the carbohydrate binding specificity.Motif QPN was mutated to QPD（Gln-Pro-Asp）and EPN（Glu-Pro-Asn）.After genetic recombination and protein expression,target protein and mutant protein were purified.In previous study,PtCTL-9,M1 and M2 displayed different PAMPs binding activities.PtCTL-9 showed a dose-dependent binding activity towards LPS,PGN,and glucan.However,M1 and M2 possessed weak binding activities towards PAMPs.Microbe binding activity showed that PtCTL-9 could bind all test microorganisms including gram-negative bacteria（V.vulnificus and V.parahaemolyticus）,gram-positive bacteria（M.luteus and S.aureus）and yeast（S.cerevisiae and P.pastoris）.The two mutated proteins M1 and M2 could not bind to these microorganisms.Microbial agglutination assay showed that PtCTL-9,M1,and M2 displayed agglutination activity towards P.pastoris in the present of 10 m M Ca²⁺.Further studies have found that PtCTL-9 can significantly inhibit the growth of S.cerevisiae.Opsonin activity research show that PtCTL-9could enhance hemocyte phagocytic activity against M.Luteus and E.coli.M2 could enhance hemocytes phagocytic activity against M.Luteus.However,M1 lost the phagocytosis enhancement activity towards M.Luteus and E.coli.Hemocytic encapsulation enhancement activity showed that r PtCTL-9,M1,and M2 could significantly enhance hemocytic encapsulation activity towards agarose.Carbohydrate binding specificity assay showed that PtCTL-9 could specifically bind to L-mannose.M1 could specifically bind to D-galactose,D-mannose,L-mannose,D-fucose,L-fucose,and D-lactose.M2 could specifically bind to L-mannose,L-fucose,and D-lactose.In conclusion,PtCTL-9 not only displayed broad non-self recognition activity,but also functioned as an opsonin to promote phagocytosis and the in vitro encapsulation activity of hemocytes.QPN motif is important in non-self recognition activity.This study provides more materials for C-type lectins in marine invertebrates and further explored the carbohydrate binding specificity of C-type lectins in invertebrates.It is helpful to carry out the relevant applied research in the future.