A multi-CRD C-type lectin with broad recognition spectrum and cellular adhesion fromArgopectenirradians

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C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins which play significant roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, a novel C-type lectin with four dissimilar carbohydrate-recognition domains (CRDs) was identified from Argopectenirradians (designated as AiCTL-9). The full-length cDNA of AiCTL-9 was of 2291 bp with an open reading frame of 1827 bp encoding a polypeptide of 608 amino acids with a signal sequence and four CRDs. The motifs determining carbohydrate binding specificity in each CRD of AiCTL-9 were different, and they were YPT in CRD1, EPD in CRD2, EPN in CRD3 and QPN in CRD4, respectively. All the four CRDs shared the similar potential tertiary structure of a typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The mRNA transcripts of AiCTL-9 were mainly detected in hepatopancreas as well as gonad, and also marginally detectable in mantle, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after the challenges of fungi PichiapastorisGS115 (P < 0.05), Gram-positive bacteria Micrococcusluteus (P < 0.05) and Gram-negative bacteria Vibrioanguillarum (P < 0.01). The recombinant AiCTL-9 (rAiCTL-9) could bind various PAMPs, including LPS, PGN, mannan and glucan, and also displayed agglutinating activity to fungi P. pastorisGS115, Gram-positive bacteria Bacillussubtilis and Gram-negative bacteria EscherichiacoliTOP10F′ as well as V. anguillarum in a Ca2+ dependent manner. Moreover, rAiCTL-9 could initiate the cellular adhesion of hemocytes and enhance their encapsulation invitro. All these results implied that AiCTL-9 was a novel PRR involved in immune response of scallop against a large number of pathogens by recognizing different PAMPs and enhancing scallop hemocytes encapsulation.

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