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-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.
D000215:Acylation; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D045804:Mycoplasma salivarium / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism*; D017354:Point Mutation; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D013211:Staphylococcus aureus / Q000378:metabolism*; D051195:Toll-Like Receptor 2; D051216:Toll-Like Receptor 6; D051193:Toll-Like Receptors; D014162:Transfection
D001426:Bacterial Proteins; D004075:Diglycerides; C460864:FSL-1 lipoprotein, synthetic; D008074:Lipoproteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009842:Oligopeptides; D010457:Peptidoglycan; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495368:TLR6 protein, human; D051195:Toll-Like Receptor 2; D051216:Toll-Like Receptor 6; D051193:Toll-Like Receptors; D007930:Leucine