Molecular characterization of a Toll-like receptor 22 homologue in large yellow croaker ( Pseudosciaena crocea) and promoter activity analysis of its 5′-flanking sequence

Abstract

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP), and induce antimicrobial immune responses. Here we report the molecular cloning and characterization of a TLR22 homologue from the large yellow croaker (LycTLR22), an economically important marine fish in China. The complete cDNA of LycTLR22 is 4607 nucleotides (nt) long, encoding a protein of 963 amino acids (aa). The deduced LycTLR22 has a typical TLR domain architecture including 18 leucine-rich repeats (LRRs) (residues 69–638) and one C-terminal LRR domain (residues 698–749) at the extracellular region, and a TIR domain (residues 805–948) in the cytoplasmic region. The three highly conserved regions among the family of bony fish were found in LycTLR22 (box 1, Y 806DAFISY 812; box 2, L 837C-RD-PG 846; box 3, W 938 surrounded by basic residue). The deduced LycTLR22 protein has 61.2%, 55.0%, 43.8%, 43.5%, 38.6% and 36.4% identity to Japanese flounder, fugu, Atlantic salmon, rainbow trout, zebrafish and goldfish TLR22, respectively. Genomic analysis showed that LycTLR22 gene consisted of four exons and three introns, which is similar to its homologue in Japanese flounder. The 5′-flanking region of LycTLR22 gene, containing several putative transcription factor binding sites and an interferon-sensitive response element (ISRE), exhibits promoter activity. LycTLR22 gene was constitutively expressed in anterior kidney, spleen, liver, heart, intestine and blood, but not in gills and muscle. Upon stimulation with poly (I:C), the LycTLR22 expression was obviously upregulated in anterior kidney and spleen tissues, and also in primary anterior kidney cells of large yellow croaker, suggesting a role for LycTLR22 in the immune response induced by poly (I:C).