CAJ | 학술논문

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用.研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义.运用基于强度的三通道荧光共振能量转移技术(FRZT)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域.结果表明:N端Glu~(24)～Met~(41)缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu~(24)～Met~(41)的TLR4不能聚合.上述结果提示,TLR4的Glu24～Met41不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用.
지다당(LPS)적식별화신호전도시숙주발생방어반응적관건,Toll양수체4(TLR4)여수양분화단백-2(MD-2)형성복합물재LPS적식별급기신호전도중발휘료중요작용.연구TLR4여MD-2결합적공능결구역,대우심입료해LPS신호전도궤제급기내독소휴극적방치구유중요의의.운용기우강도적삼통도형광공진능량전이기술(FRZT)급기인돌변화전염기술,연구료활세포TLR4여MD-2작용적결구역.결과표명:N단Glu~(24)～Met~(41)결실사TLR4여MD-2결합능력명현하강;LPS자격후TLR4취합신속증가,이결실Glu~(24)～Met~(41)적TLR4불능취합.상술결과제시,TLR4적Glu24～Met41불부시결합MD-2적구역,병차환삼여료LPS자격후TLR4적취합작용.
TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.