Molecular and functional characterization of Toll-like receptor 21 in large yellow croaker (Larimichthys crocea)

Toll-like receptor 21 (TLR21) is a non-mammalian TLR that functions similar to mammalian TLR9 in recognizing CpG DNA. In the present study, we identified a TLR21 homologue, LycTLR21, from large yellow croaker (Larimichthys crocea). The complete coding sequence of LycTLR21 is 2946 nucleotides long, encoding a protein of 981 amino acids. The deduced LycTLR21 protein has typical TLR domain architecture, including a signal peptide, 13 leucine-rich repeats (LRRs) in the extracellular region, a transmembrane region, and a cytoplasmic Toll-Interleukin-1 receptor (TIR) domain. Phylogenetic analysis showed that LycTLR21 falls into a major clade formed by all fish TLR21 sequences and is closely related to TLR21 in Epinephelus coioides and Oplegnathus fasciatus. LycTLR21 mRNA was constitutively expressed in all tissues tested, with higher levels in immune-related tissues, such as spleen, head kidney, and gills. Upon stimulation with inactivated trivalent bacterial vaccine, LycTLR21 mRNA was significantly increased in these three tissues. Overexpression of a chimeric plasmid containing the extracellular domain of human cluster of differentiation 4 (CD4) and the transmembrane and cytoplasmic domains of LycTLR21 could activate NF-κB, but not IFN-β in Chinese hamster ovary (CHO) cells, suggesting that LycTLR21 could mediate activation of NF-κB. LycTLR21 could specifically recognize three CpG-oligodeoxynucleotides (CpG-ODNs), CpG-ODN 1826, 2006, and 2007, but not other CpG-ODNs detected, poly(I:C), lipopolysaccharide (LPS), and lipoteichoic acid (LTA-SA). These three CpG-ODNs were found to significantly up-regulate the expression of LycTLR21 and downstream proinflammatory cytokines IL-1β and IL-6 of NF-κB pathway in large yellow croaker head kidney (LYCK) cells. In addition, the expression levels of LycTLR21, c-Rel subunit of NF-κB, IL-1β and IL-6 genes were quickly increased in the spleen and head kidney by bacterial infection, suggesting that LycTLR21 signaling pathway may play a role in immune response to bacterial infection.