Kiyokawa Takashi, Akashi-Takamura Sachiko, Shibata Takuma, Matsumoto Fumi, Nishitani Chiaki, Kuroki Yoshio, Seto Yasuyuki, Miyake Kensuke
Division of Infectious Genetics, The Institute of Medical Science, Tokyo, Japan.
Int Immunol. 2008 Nov;20(11):1407-15. doi: 10.1093/intimm/dxn098. Epub 2008 Sep 9.
Toll-like receptors (TLRs) play an essential role in defense responses. Immune cells express multiple TLRs which are simultaneously activated by microbial pathogens. PRotein Associated with Tlr4 A (PRAT4A) is a chaperone-like endoplasmic reticulum (ER)-resident protein required for the proper subcellular distribution of multiple TLRs. PRAT4A(-/-) mice show impaired expression of TLR2/4 on the cell surface and the lack of ligand-induced TLR9 relocation from the ER to endolysosome. Consequently, TLR responses to whole bacteria as well as to TLR2, 4 and 9 ligands are impaired. We here compare the interaction of these TLRs with PRAT4A. Association of endogenous PRAT4A was easily detected only with TLR4. The TLR4 region responsible for strong interaction with PRAT4A is very close to the site necessary for interaction with MD-2. By using transient expression, we were able to detect PRAT4A interaction with TLR2 and TLR9. The PRAT4A single-nucleotide mutant replacing methionine 145 with lysine (M145K) associates with TLR9 but does not rescue ligand-dependent TLR9 trafficking. By contrast, the M145K mutant weakly, if at all, associates with TLR2 and TLR4. The M145K mutant appreciably rescues cell-surface TLR2 expression and its responses in PRAT4A(-/-) bone marrow-derived dendritic cells, whereas little if any rescue of cell-surface TLR4/MD-2 expression and its responses occurs. These results demonstrate that PRAT4A differentially interacts with each TLR and suggest that a single-nucleotide change in the PRAT4A gene influences not only the strength of TLR responses but can also alter the relative activity of each TLR.
Toll样受体（TLR）在防御反应中发挥着至关重要的作用。免疫细胞表达多种TLR，它们会被微生物病原体同时激活。与Tlr4相关的蛋白A（PRAT4A）是一种类似伴侣蛋白的内质网（ER）驻留蛋白，是多种TLR正确亚细胞分布所必需的。PRAT4A基因敲除（-/-）小鼠在细胞表面的TLR2/4表达受损，并且缺乏配体诱导的TLR9从内质网向内溶酶体的重新定位。因此，TLR对全细菌以及对TLR2、4和9配体的反应均受损。我们在此比较了这些TLR与PRAT4A的相互作用。仅与TLR4容易检测到内源性PRAT4A的结合。与PRAT4A发生强烈相互作用的TLR4区域非常接近与MD-2相互作用所需的位点。通过瞬时表达，我们能够检测到PRAT4A与TLR2和TLR9的相互作用。用赖氨酸替代甲硫氨酸145的PRAT4A单核苷酸突变体（M145K）与TLR9结合，但不能挽救配体依赖性的TLR9转运。相比之下，M145K突变体与TLR2和TLR4的结合较弱（如果有结合的话）。M145K突变体可明显挽救PRAT4A基因敲除（-/-）骨髓来源的树突状细胞中细胞表面TLR2的表达及其反应，而对细胞表面TLR4/MD-2的表达及其反应几乎没有挽救作用。这些结果表明，PRAT4A与每种TLR的相互作用存在差异，并表明PRAT4A基因中的单核苷酸变化不仅会影响TLR反应的强度，还会改变每种TLR的相对活性。
