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MD-2 依赖性人 Toll 样受体 4 单克隆抗体检测细胞表面 Toll 样受体 4 与外在可溶性 MD-2 的细胞外关联。

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.

机构信息

Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Sendai 980-8578, Japan; Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan.

出版信息

Biochem Biophys Res Commun. 2013 Oct 11;440(1):31-6. doi: 10.1016/j.bbrc.2013.09.004. Epub 2013 Sep 8.

Abstract

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.

摘要

MD-2 对于 Toll 样受体 4(TLR4)识别脂多糖(LPS)是必不可少的,但对于细胞表面表达则不是。TLR4/MD-2 复合物是通过共表达在细胞内形成的。由于在没有 TLR4 共表达的情况下分泌的 MD-2 具有聚集性质,因此细胞外复合物的形成仍然存在争议。我们使用三种独立的单克隆抗体(mAb)证明了细胞外复合物的形成,这三种 mAb 均特异性针对复合物化的 TLR4,但与游离的 TLR4 和 MD-2 无反应。这些 mAb 仅在与分泌 MD-2 的中国仓鼠卵巢细胞共培养或与这些细胞的条件培养基孵育时才与表达 TLR4 的 Ba/F3 细胞结合。在滴定研究中,所有三种 mAb 均能与细胞外形成的复合物结合,而不能与细胞内形成的复合物结合。此外,我们证明两种 mAb 在 LPS 刺激下失去了与 TLR4/MD-2 的亲和力,这表明这些 mAb 结合了构象敏感表位。在 LPS 刺激下,也发现了这种情况。此外,我们表明细胞表面 TLR4 和外在分泌的 MD-2 能够在细胞外形成功能性复合物,这表明复合物形成存在另一种或替代途径。

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