A C-type lectin from Crassostrea gigas with novel EFG/FVN motif involved in recognition of various PAMPs and induction of interleukin expression.

Abstract

C-type lectins (CTLs) are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which participate in the nonself-recognition and triggering the transduction pathways in the innate immunity. In the present study, a novel CTL (designated as CgCLEC-TM2) with a carbohydrate-recognition domain (CRD) and a transmembrane domain (TM) was identified from the Pacific oyster Crassostrea gigas. Two novel EFG and FVN motifs were found in Ca2+-binding site 2 of CgCLEC-TM2. The mRNA transcripts of CgCLEC-TM2 were detected in all tested tissues with the highest expression level in haemocytes, which was 94.41-fold (p<0.01) of that in adductor muscle. The relative expression level of CgCLEC-TM2 in haemocytes significantly up-regulated at 6h and 24h 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 CgCLEC-TM2 (rCRD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN), and poly (I: C) in a Ca2+-dependent manner. The rCRD exhibited binding activity to V. anguillarum, Bacillus subtilis, V. splendidus, Escherichia coli, Pichia pastoris, Staphylococcus aureus and Micrococcus luteus in a Ca2+-dependent manner. The rCRD also exhibited agglutination activity to E. coli, V. splendidus, S. aureus, M. luteus and P. pastoris in a Ca2+-dependent manner. The phagocytosis rate of haemocytes towards V. splendidus significantly down-regulated from 27.2% to 20.9% after treatment of anti-CgCLEC-TM2-CRD antibody, while the growth of V. splendidus and E. coli was inhibited compared with the TBS and rTrx groups. After the expression of CgCLEC-TM2 was inhibited by RNAi, the expression level of phospho-extracellular regulated protein kinases (p-CgERK) in haemocytes, and the mRNA expressions of interleukin17s (CgIL17-1 and CgIL17-4) decreased significantly after V. splendidus stimulation, compared with that in EGFP-RNAi oysters, respectively. These results suggested that CgCLEC-TM2 with novel motifs served as a pattern recognition receptor (PRR) involved in the recognition of microorganisms, and induction of CgIL17s expression in the immune response of oysters.