Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea)

Abstract

The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

Keywords: C-type lectin-like receptor; antibacterial immunity; bacterial agglutination; hemagglutination; large yellow croaker Larimichthys crocea.

Figure 1

Alignment of deduced amino acid sequences of LycCTLR with other species CTLR, DC-SIGN, and L-SIGN molecules. Sequence alignments were obtained with ClustalX2 by the ClustalW method and conserved residues are shaded using BOXSHADE (v3.21, Swiss institute of bioinformatics, Swaziland). The transmembrane domain is boxed. The C-type lectin-like domain is underlined. The identical residues are indicated with black background, and similar residues are indicated with dark grey background. The six highly conserved cysteine residues are indicated by arrows. EPD motif and WYD motif are indicated with solid and hollow cycles below, respectively. Ca²⁺ binding site 1 are marked with solid diamond. Ca²⁺ binding site 2 are marked with hollow diamond.

Figure 2

Phylogenetic tree based on the genetic distances of deduced amino acid sequences of fish CTLR, DC-SIGN, and L-SIGN. Deduced amino acid sequences of fish CTLR, DC-SIGN, and L-SIGN were aligned using CLUSTAL X, and the tree was constructed with the neighbour-joining method by MEGA 6.0 software [19]. Numbers on nodes represent frequencies with which the node is recovered per 100 bootstrap replications in a total of 10,000. The LycCTLR is boxed and in bold. The GenBank accession numbers of the sequences used here are shown in the figure.

Figure 3

Genomic structure analyses of CTLR and DC-SIGN genes from fish and mammals. Exons are represented by closed boxes, and introns are represented by horizontal lines. Open boxes indicate UTR regions. The nucleotide length is shown in boxes and above lines. The genomic DNA sequences (and their accession numbers) are taken from GenBank database: CTLR: large yellow croaker, KQ041363 (EH28_17225); tilapia, NC_022209; guppy, NC_024346; salmon A, NM_001123579; spotted gar, NC_023204; mouse, NC_000072.6; human, AC092746.9. DC-SIGN: large yellow croaker, KQ041008; DC-SIGN-like, KQ041102; zebrafish, NC_007121.6; human, NC_000019.10; mouse, NC_000074.6.

Figure 4

Expression analysis of LycCTLR gene in various tissues. Total RNA was extracted from eight tissues each from four normal fish, respectively, and RT-PCR was used to detect the expression levels of LycCTLR in various tissues. As a positive control for RT-PCR, β-actin was amplified to determine the concentration of templates. M: DNA Marker; 1. Gills; 2. Intestine; 3. Liver; 4. Kidney; 5. Heart; 6. Spleen; 7. Muscle; 8. Blood.

Figure 5

Expression analysis of LycCTLR gene in head kidney (A) and spleen (B) after inactivated trivalent bacterial vaccine induction. Head kidney and spleen were collected at 0, 12, 24, 48, and 72 h after bacterial vaccine induction, and total RNA was extracted for real-time PCR analysis. The relative expression level of LycCTLR was normalized by that of β-actin. The fold change was calculated as the average expression level of LycCTLR in the bacterial vaccine-challenged samples divided by that in the samples from phosphate buffered saline (PBS)-injected fish at each time point. Each experiment was performed in triplicate; error bars represent the standard error of the mean (SEM). The statistical significance of differences in gene expression was generated by two-tailed Student’s t-test compared with the data from PBS-injected group (* p < 0.05; ** p < 0.01).

Figure 6

Bacterial agglutination caused by recombinant LycCTLR protein. For bacterial agglutination assays, eight strains of bacteria mentioned in the Materials and methods were tested. The results showed that the rLycCTLR at 64 μg/mL could agglutinate Gram-negative bacterium E. coli and Gram-positive bacterium B. cereus, but not the other six bacterial strains. No agglutinating activity was observed in the rLycCTLR without Ca²⁺ group and rLycactin control. The scale bar shown in the lower right is 200 μm.