Characterization of bovine Toll-like receptor 8: ligand specificity, signaling essential sites and dimerization.
Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors (PRRs) and are essential for host immune response. Little is known regarding the activation mechanism of TLRs especially of the TLR7/8/9 subfamily. Here we cloned and characterized bovine TLR8 (bTLR8) and found that it is highly responsive to two TLR7 ligands, imiquimod and gardiquimod, in transfected cell lines. Using the transfected cell lines as model systems, we analyzed by mutagenesis the roles of potentially important regions of bTLR8 in receptor signaling: 5 insertions in leucine rich repeats (LRRs) of the ectodomain (ECD), 9 N-glycosylation sites, all the cysteines, an aspartate conserved between TLRs, the transmembrane (TM) domain and different cytoplasmic regions. All 5 insertions, 2 N-glycosylation sites, most of the cysteines, the conserved aspartate, the TM and each of the cytoplasmic regions are essential for TLR8 signaling. We also showed that bTLR8 undergoes dimerization/self-association which was not affected by imidazoquinoline stimulation. This observation together with kinetics of activation suggested that a ligand-induced dimer conformational switch is mainly responsible for TLR8 activation. All the TLR8 signaling essential sites were examined for their requirement in dimerization; no single mutation or group of mutations affected the dimerization. However, among the impaired TLR8 mutants, all those containing mutations in the transmembrane or cytoplasmic regions and only two within the ECD (N515D and D536A) showed dominant negative inhibition to wild type receptor, whereas the others, all within the ECD, did not compete with wild type TLR8. A model for activation of bTLR8 was described based on these data.
Zhu
Jianzhong
J
Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3.
Brownlie
Robert
R
Liu
Qiang
Q
Babiuk
Lorne A
LA
Potter
Andrew
A
Mutwiri
George K
GK
D000818:Animals; D019556:COS Cells; D002417:Cattle / Q000235:genetics / Q000276:immunology*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019281:Dimerization; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016296:Mutagenesis; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D051199:Toll-Like Receptor 7 / Q000235:genetics / Q000276:immunology; D051200:Toll-Like Receptor 8 / Q000235:genetics / Q000276:immunology*; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000276:immunology
D008024:Ligands; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D051217:Toll-Like Receptor 9