Abstract:
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-
Clec4
e (C-type lectin domain family 4 member E gene,
Clec4
e), 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
LcClec4
e 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
LcClec4
e was closely related to CTLRs of a variety of fishes of the order Perciformes. The results of real-time quantitative PCR showed that
LcClec4
e was constitutively distributed in the 10 tissues tested, and the expression level was the highest in the liver;
LcClec4
e 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
LcClec4
e in these 3 kinds of immune cells were significantly upregulated after stimulation by inactivated
Vibrio alginolyticus. The extracellular segment of
LcClec4e (r
LcClec4e-extracellular domain, r
LcClec4e-ex) expressed in
Escherichia coli BL21 had Ca
2+-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 r
LcClec4e-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.