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Abstract
Synthetic phosphorothiolate-modified CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli. Toll-like receptor (TLR) 9 and TLR21 are their cellular receptors in different species. The structural requirements for CpG-ODN to strongly activate TLR9 have been relatively well studied, but studies on TLR21 are in their infancy. Therefore, in this study, we investigated the interaction between CpG-ODNs and TLR21s from groupers (Epinephelus spp.), which are economically important fish species. We cloned the cDNA of giant grouper (E. lanceolatus) TLR21, and compared its sequence with orange-spotted grouper (E. coioides) TLR21A and TLR21B. These three receptors were activated by CpG-ODNs containing the GTCGTT motif but not by those containing the GACGTT motif. We developed two CpG-ODNs that contained 19 phosphorothiolated deoxynucleotides with one or two GTCGTT motifs. These CpG-ODNs had better activity on grouper TLR21s than currently developed CpG-ODNs, and produced similar immune stimulatory profiles when applied to cells isolated from orange-spotted grouper. The developed CpG-ODNs also effectively activated both human and mouse TLR9-mediated NF-κB activation and cytokine productions. These findings suggest that the GTCGTT motif is required for CpG-ODNs to activate grouper TLR21s, and that the CpG-ODNs that were developed for grouper TLR21s contain structures that effectively activate human and mouse TLR9s.
Highlights
Synthetic phosphorothiolate-modified CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli
The developed CpG-ODNs effectively activated both human and mouse TLR9-mediated nuclear factor (NF)-κB activation and cytokine productions. These findings suggest that the GTCGTT motif is required for CpG-ODNs to activate grouper TLR21s, and that the CpG-ODNs that were developed for grouper TLR21s contain structures that effectively activate human and mouse TLR9s
The main difference between their encoded protein sequences is the absence of four amino acid residues at the C-terminal end of osgTLR21B (Supplementary Fig. 1)
Summary
Synthetic phosphorothiolate-modified CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli. We cloned the cDNA of giant grouper (E. lanceolatus) TLR21, and compared its sequence with orange-spotted grouper (E. coioides) TLR21A and TLR21B These three receptors were activated by CpG-ODNs containing the GTCGTT motif but not by those containing the GACGTT motif. We developed two CpG-ODNs that contained 19 phosphorothiolated deoxynucleotides with one or two GTCGTT motifs These CpG-ODNs had better activity on grouper TLR21s than currently developed CpG-ODNs, and produced similar immune stimulatory profiles when applied to cells isolated from orange-spotted grouper. Most of our current knowledge about the structural–functional relationships and species-specific activities of CpG-ODNs is generated by studying their interaction with TLR9, but much less is derived from the study with TLR21 This is because most of the earlier work on CpG-ODNs was performed using human and mouse cells, which contain only TLR9. The sequence that is required to strongly activate TLR21 has not been investigated and it remains unclear whether a CpG-ODN can simultaneously have strong activity toward both TLR9 and TLR21
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