Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2 ^ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser⁴⁰ to Ile⁶⁴) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2^ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu¹⁷⁸ to Ala (TLR2^E178A), TLR2 ^E180A, TLR2^E190A, and TLR2^L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2^L107E, TLR2 ^L112E (a TLR2 point mutant with a substitution of Leu¹¹² to Glu), and TLR2^L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2^L115E, TLR2 ^L112E, and TLR2^ΔS40-164 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2^E190A were. In addition, these mutants, except for TLR2^E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser⁴⁰-Ile⁶⁴ and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.