Furthermore, we have identified two regions in the ectodomain of TLR9 as important for receptor activation.Replacing amino acid residues Gln346, Arg348 and Gln562 of the human TLR9 to mouse counterparts increase activation of TLR9 with mouse specific ODNs, bacterial DNA and mammalian genomic DNA. We proposed that bias for double CpG motif of human TLR9 results in decreased activation by the DNA that has a lower incidence of CpG motifs, such as the mammalian genomic DNA. The findings are important for understanding the mechanism of activation of TLR9 receptor and design of new synthetic agonists as potential drugs.
COBISS.SI-ID: 6089754
Natural activators of receptor TLR9 are bacterial and viral DNA. In addition to microbial DNA the TLR9 is activated by DNA of the host, which is released upon trauma and can trigger TLR9-mediated sterile inflammation. Degraded DNA is a source of DNA fragments, of which only single-stranded DNA with corresponding length and unmethylated CpG motif activates TLR9. We have shown that short DNA (2-5 nt), which contains a CG dinucleotide (sODN), binds to TLR9, but on its own do not induce activation of TLR9. However, in the presence of synthetic agonists sODNs significantly augment activation of TLR9. sODNs enhance TLR9 activation by bacterial DNA and methylated mammalian genomic DNA. The synergistic activities of sODNs are even more pronounced in the presence of TLR9 ligands, which by themselves fail to activate the receptor. We have shown that short DNA fragments are generated by degradation of genomic DNA and are internalized by endocytosis. These results show the role of DNA degradation products in association with infections, or autoimmune diseases, essentially at the limiting quantities agonists.
COBISS.SI-ID: 6146842
Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. We report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.
COBISS.SI-ID: 39287301
The systematic analysis of species and sequence specific activation of TLR9 was published in three closely-related publications in the Journal of Immunology. The CpG-containing oligodeoxyribonucleotides (ODNs) activate endosomal transmembrane TLR9 receptor; however, their activation efficiency depends on length, nucleotide sequence and dimerization properties. The systematic analysis of sequence and functional features of B-type agonists revealed important differences between minimal sequence requirements for activation of human and mouse TLR9. We demonstrated with a systematic investigation of the sequence motifs of phosphodiester oligonucleotides (ODNs) activating receptor TLR9 that ODNs shorter than 21 nt and with the adenosine adjacent to the cytidine-guanosine (CG) dinucleotide motif led to a significant loss of the propensity to activate TLR9. The distance between the stimulatory CpG motifs within the ODN fine-tunes the activation of B cells. The minimal ODNs that activate human TLR9 comprise 2 CG dinucleotides separated by 6–10 nt, where the first CpG motif is preceded by the 5′-thymidine and the elongated poly-thymidine tail at the 3′ end of the ODN. The minimal sequence provides insight into the molecular mechanism of TLR9 ligand recognition
COBISS.SI-ID: 37867781
The CpG-containing oligodeoxyribonucleotides (ODNs) activate endosomal transmembrane TLR9 receptor; however, their activation efficiency depends on length, nucleotide sequence and dimerization properties. The systematic analysis of sequence and functional features of B-type agonists revealed important differences between minimal sequence requirements for activation of human and mouse TLR9. For activating mouse TLR9 a single CpG motif positioned 4–6 nt from the 5’-end is sufficient. The 5 'TCC motif 1-3 nt away of a CpG motif augment activation of TLR9. The distance of the CG dinucleotide of 4 to 6 nt from the 5’-end and the ODN’s length fine-tunes activation of mouse macrophages. Identification of the minimal sequence provides an insight into the sequence selectivity of mouse and human TLR9 and points to the differences in the receptor selectivity between species probably as a result of differences in the receptor binding sites.
COBISS.SI-ID: 5796378