COMPARATIVE STUDY
JOURNAL ARTICLE
RESEARCH SUPPORT, NON-U.S. GOV'T
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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.

Journal of Immunology 2003 October 2
S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-kappaB 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(DeltaS40-I64) (a TLR2 mutant with a deletion of the region of Ser(40) to Ile(64)) failed to activate NF-kappaB in response to FSL-1. The deletion mutant TLR2(DeltaC30-S39) induced NF-kappaB 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 Glu(178) to Ala (TLR2(E178A)), TLR2(E180A), TLR2(E190A), and TLR2(L132E) induced NF-kappaB 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 Leu(112) to Glu), and TLR2(L115E) failed to induce NF-kappaB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2(L115E), TLR2(L112E), and TLR2(DeltaS40-I64) 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 Ser(40)-Ile(64) 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.

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