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

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Abstract
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 TLR2ΔS40-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 TLR2ΔC30-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 (TLR2E178A), TLR2E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2L112E, and TLR2ΔS40-I64 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, 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.