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
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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DOI：
10.4049/jimmunol.171.7.3675
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发表时间：
2003-10-01
影响因子：
4.4
通讯作者：
Shibata, K
中科院分区：
文献类型：
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作者：
Fujita, M;Into, T;Shibata, K
唾液支原体来源的S-(2，3-bispaimitoirioxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe(FsL-1)对Toll样受体2(TLR2)或TLR2和TLR6共转染的人胚胎肾2 93(HEK2 93)细胞的核转录因子-kappaB报告基因活性有明显的促进作用，但对TLR6的HEK2 93细胞无明显影响。同时转导TLR2和TLR6的HEK293细胞的活性明显高于仅转导TLR2的HEK293细胞。缺失突变体TLR2(DeltaS40-164)(缺失Ser(40)~Ile()区域的TLR2突变体)未能激活NF-kappaB以响应FSL-1。缺失突变体TLR2(DeltaC30-S39)可诱导NF-kappaB报告基因的活性，但与野生型TLR2相比，其活性水平显著降低。在FSL-1、唾液分枝杆菌脂蛋白和金黄色葡萄球菌肽聚糖的刺激下，TLR2(178)、TLR2(E180A)、TLRE190A和TLR2(L132E)均不能诱导NF-kappaB的激活，但TLR2L(107E)、TLR2(L112E)(TLR2(L112E)(TLR2点突变体，Leu(112)替换为Glu)和TLR2(L115E)不能诱导NF-kappaB的激活，提示这些残基对它们的信号转导是必不可少的。流式细胞仪分析表明，TLR2(L115E)、TLR2(L112E)和TLR2(DeltaS40-164)与野生型TLR2(E190A)一样在细胞表面表达。此外，除TLR2(E180A)外，这些突变体都是TLR2的显性负型。本研究强烈提示TLR2富亮氨酸重复序列中的Ser(40)-Ile()胞外区和第107、112、115位亮氨酸残基参与了对支原体二酰化脂蛋白和脂肽的识别以及对金黄色葡萄球菌肽多聚糖的识别。
S-(2,3-bispaimitoirioxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasnia 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(DeltaS40-164) (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(E178)), TLR2(E180A) TLRE190A, and TLR2 (L132E) induced NF-kappaB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Stapikylococcus aureus peptidoglycans, but TLR2L(107E) 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(DeltaS40-164) 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 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.