Molecular identification and agglutination characteristics of C-type lectin receptor Clec4e in large yellow croaker (Larimichthys crocea)

C-type lectin receptor (CTLR) is a kind of pattern recognition receptors that can specifically bind to carbohydrates pathogen-associated molecular patterns (PAMPs) and plays an important role in innate immunity. In order to elucidate the biological function of CTLR in teleost fish, a CTLR gene-Clec4e (C-type lectin domain family 4 member E gene, Clec4e), which was screened from the transcriptome database of large yellow croaker (Larimichthys crocea), was taken as the object of the present study, and its molecular features, expression distribution and agglutination characteristics were studied. The results showed that the full-length cDNA of LcClec4e was 1 546 bp, with an open reading frame (ORF) of 771 bp, encoding 254 amino acids. The N-terminus of LcClec4e had a transmembrane region without a signal peptide, and the C-terminus had a carbohydrate recognition domain (CRD), which contained carbohydrate-binding sites EPN and WFD and 6 conserved cysteines what could form disulfide bonds. Phylogenetic analysis showed that LcClec4e was closely related to CTLRs of a variety of fishes of the order Perciformes. The results of real-time quantitative PCR showed that LcClec4e was constitutively distributed in the 10 tissues tested, and the expression level was the highest in the liver; LcClec4e was expressed in primary macrophages, lymphocytes and granulocytes from the head kidney tissues of L. crocea, and the expression level was the highest in macrophages; the expression levels of LcClec4e in these 3 kinds of immune cells were significantly upregulated after stimulation by inactivated Vibrio alginolyticus. The extracellular segment of LcClec4e (rLcClec4e-extracellular domain, rLcClec4e-ex) expressed in Escherichia coli BL21 had Ca²⁺-dependent agglutination activity and could agglutinate mouse, rabbit erythrocytes and 4 Gram-negative bacteria common in aquaculture, such as Aeromonas hydrophila, Pseudomonas plecoglossicida, V. alginolyticus, V. campbellii. D-glucose, D-fructose, D-mannose, D-maltose, α-lactose, and lipopolysaccharide all had inhibitory effects on the agglutination of rLcClec4e-ex toward P. plecoglossicida, suggesting that LcClec4e could bind to carbohydrates of P. plecoglossicida. The results above indicated that LcClec4e might be an PPR which could recognize bacteria by binding to carbohydrates PAMPs, thus involved in the immune defense of L. crocea against bacterial infection.