Replacement of two aminoacids in the bovine Toll-like receptor 5 TIR domain with their human counterparts partially restores functional response to flagellin

• We compared and modelled TLR5 TIR domain sequences.
• AA substitutions in boTLR5 TIR domain leads to absence of PI3K phosphorylation site.
• Exchange of AA with corresponding huTLR5 AA partially restores functionality.
• This substitution does not seem to impact on PI3K phosphorylation in boTLR5.

Flagellin potently induces inflammatory responses in mammalian cells by activating Toll-like receptor (TLR) 5. Recently, we were able to show that stimulation of bovine TLR5 resulted in neither NFκB signalling nor CXCL8 production. Like other TLRs, TLR5 recruits signalling molecules to its intracellular TIR domain, leading to inflammatory responses. Analysis of available TLR5 sequences revealed substitutions in all artiodactyl sequences at amo acid (AA) position 798 and 799. Interestingly, a putative binding site for PI3K was identified at tyrosine 798 in the human TLR5 TIR domain, analogous to the PI3K recruitment domain in the IL-1 receptor. Mutation of the artiodactyl residues at position 798, 799 or both with their corresponding human counterparts partially restored the response of bovine (bo)TLR5 to flagellin as well as phosphorylation of PI3K. Together, our results suggest a potential lack of phosphorylation of F798 and H799 in boTLR5 partially explains the lack in observed response.