Leucine-rich Repeat 11 of Toll-like Receptor 9 Can Tightly Bind to CpG-containing Oligodeoxynucleotides, and the Positively Charged Residues Are Critical for the High Affinity
Leucine-rich Repeat 11 of Toll-like Receptor 9 Can Tightly Bind to CpG-containing Oligodeoxynucleotides, and the Positively Charged Residues Are Critical for the High Affinity
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Toll样受体9富含亮氨酸的重复序列11可以与含CpG的寡脱氧核苷酸紧密结合，带正电荷的残基对于高亲和力至关重要
DOI：
10.1074/jbc.m112.396432
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发表时间：
2012-08-31
影响因子：
4.8
通讯作者：
Zhou, Hong
中科院分区：
文献类型：
--
作者：
Pan, Xichun;Yue, Junjie;Zhou, Hong
TLR 9是一种感受细菌DNA/含CpG寡核苷酸（CpG ODN）的受体。人TLR 9（hTLR 9）的胞外结构域（ECD）由25个富含亮氨酸的重复序列（LRR）组成，有助于CpG ODN的结合。在此，我们发现在LRR 2、-5、-8和-11中，hTLR 9的LRR 11对CpG ODN具有最高的亲和力，其次是LRR 2和-5，而LRR 8几乎没有亲和力。在体外，与LRR 2和LRR-5相比，LRR 11预孵育更显著地降低了CpG ODN处理的小鼠腹腔巨噬细胞的CpG ODN内化，随后的NF-κ B活化和细胞因子释放。hTLR 9的LRR 11缺失突变体使细胞对CpG ODN的反应降低。LRR 11（LRR 11 m1 -9）的5个正电荷残基的单位点或多位点突变，尤其是Arg-337和Lys-367，有助于hTLR 9与CpG ODN的结合。LRR 11 m1 -9对CpG ODN内化和CpG ODN/TLR 9信号转导的抑制作用减弱，支持上述发现。对hTLR 9 ECD-CpG ODN相互作用的预测表明，Arg-337和Lys-338通过氢键直接与CpG ODN结合，而Lys-347、Arg-348和His-353则有助于稳定配体结合区的形状。这些结果表明，尽管LRR 11中所有5个带正电荷的残基都有助于其高亲和力，但只有Arg-337和Lys-338直接与CpG ODN相互作用。总之，结果表明LRR 11可以与CpG ODN强烈结合，而5个正电荷残基的突变降低了这种高亲和力。LRR 11可作为hTLR 9的拮抗剂进一步研究。
TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-kappa B activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.