Comparative Analysis of Species-Specific Ligand Recognition in Toll-Like Receptor 8 Signaling: A Hypothesis

Abstract

Toll-like receptors (TLRs) play a central role in the innate immune response by recognizing conserved structural patterns in a variety of microbes. TLRs are classified into six families, of which TLR7 family members include TLR7, 8, and 9, which are localized to endolysosomal compartments recognizing viral infection in the form of foreign nucleic acids. In our current study, we focused on TLR8, which has been shown to recognize different types of ligands such as viral or bacterial ssRNA as well as small synthetic molecules. The primary sequences of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the TLR8 pathway is species-specific. Moreover, the factors underlying the receptor's species-specificity remain unknown. To this end, comparative homology modeling, molecular dynamics simulations refinement, automated docking and computational mutagenesis studies were employed to probe the intermolecular interactions between this anti-viral compound and TLR8. Furthermore, comparative analyses of modeled TLR8 (rodent and non-rodent) structures have shown that the variation mainly occurs at LRR14-15 (undefined region); hence, we hypothesized that this variation may be the primary reason for the exhibited species-specificity. Our hypothesis was further bolstered by our docking studies, which clearly showed that this undefined region was in close proximity to the ligand-binding site and thus may play a key role in ligand recognition. In addition, the interface between the ligand and TLR8s varied depending upon the amino acid charges, free energy of binding, and interaction surface. Therefore, our current work provides a hypothesis for previous in vivo studies in the context of TLR signaling.

RNA and Imidazoquinolines Are Sensed by Distinct TLR7/8 Ectodomain Sites Resulting in Functionally Disparate Signaling Events

“…HEK293T cells in 24-well plates (7.5 × 10^4 cells/well) were transfected using CaPO 4 (13) with a firefly luciferase reporter (Stratagene), Renilla luciferase control reporter (pRL-Tk), and pC1-EGFP (BD Clontech) and 24h later stimulated with 12 μg/ml DOTAP-complexed RNA40 or indicated concentrations of R848 (usually 0.75 μg/ml for TLR7 and 2.5 μg/ml for TLR8) for 18 h. Luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) on a Fluostar Optima Instrument (BMG Labtech). Mean values of triplicates (+/− SD) are shown throughout.…”

“…On the sequence level, TLR7 and TLR8 are most closely related to TLR9 and TLR3; hence an RNA recognition mechanism similar to TLR9 and TLR3 appears likely. The small size of imidazoquinolines has led to the proposal that their recognition by TLR7 and TLR8 may be altogether different to that of RNA ORN and involve insertion of a single imidazoquinoline molecule into a small binding interface (13). However, until recently, no systematic studies addressing the recognition principles for RNA ORN and imidazoquinolines by TLR7/8 have been conducted.…”

Cell Type-Specific Responses of Peripheral Blood CD14-Positive Monocytes to Liposome-Encapsulated Immunostimulatory siRNA

“…TLR8 is phylogenetically and structurally similar to TLR7, however, there is species-specific difference in single-stranded RNA recognition. [18][19][20] For example, the imidazoquinoline resiquimod activated human TLR7, TLR8 and murine TLR7, but not murine TLR8.…”

Recognition of pathogen-associated nucleic acids by endosomal nucleic acid-sensing toll-like receptors

“…In addition, guanine analogs, such as 7-allyl-8-oxoguanosine (loxoribine) and gardiquimod, and pyrimidine analog bropirimine can induce the production of various cytokines via TLR7 in humans and mice [139,140], suggesting the possibility of exploiting imidazoquinolines and nucleoside analogs as adjuvants for therapy, vaccination, and anti-tumor response in humans and animals.…”

“…1 and Table 2). Human TLRs 7 and 8, and mouse TLR7 recognize imidazoquinolines and initiate immune response, whereas mouse TLR8 is thought not to be activated by imidazoquinolines or ssRNA due to a five amino-acid deletion in the ECD [139,140]. Nevertheless, mouse TLR8 could be activated by poly(dT)17 oligodeoxyribonucleotides (ODNs) combined with the 3M-002, leading to the induction of TNF-a [141], suggesting that mouse TLR8 may be functionally active in the detection of DNA viruses.…”