Multimeric MD-2 as an LPS decoy

描述(申请人提供)：多聚体MD-2作为内毒素诱饵识别革兰氏阴性细菌脂多糖(LPS)是由Toll样受体4(TLR4)、MD-2和CD14组成的细胞表面受体复合体介导的。MD-2是一种分泌蛋白，与TLR4的胞外部分相互作用，并能够直接与内毒素结合。MD-2既以单体形式存在，也以二硫键连接的多聚体形式存在。我们发现只有MD-2单体与TLR4结合。此外，只有MD-2单体对表达TLR4的细胞株具有内毒素反应性。因此，单体MD-2是TLR4识别内毒素的功能伙伴，而MD-2多聚体的功能尚不清楚。目前的拨款提案将检验MD-2多聚体可以充当内毒素诱饵的假设。为了证明这一性质，我们必须证明多聚体MD-2，尽管它不能与TLR4结合并赋予内毒素反应性，但仍然能够结合内毒素。具体来说，我们将：1.用生化和生物物理方法检测MD-2多聚体与内毒素的直接结合能力。2.分析纯化的MD-2多聚体加入培养的人树突状细胞、内皮细胞和表达TLR4/MD-2的细胞系中是否抑制其对内毒素的反应。3.研究促炎和抗炎刺激对MD-2‘S表达和多聚体形成的调节作用。这些研究将加深我们对内毒素识别和细胞激活的分子方面的理解，并将表征抑制内毒素作用的机制，这些机制可能被用于治疗干预。

DESCRIPTION (provided by applicant): Multimeric MD-2 as an LPS decoy Recognition of Gram-negative bacteria lipopolysaccharide (LPS) is mediated by a cell surface receptor complex constituted by Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4 and that is able to bind directly to LPS. MD-2 exists in both monomeric form and as disulfide-linked multimer. We found that only MD-2 monomer binds to TLR4. Moreover, only MD-2 monomer confers LPS responsiveness to TLR4 expressing cell lines. Thus, monomeric MD-2 is a functional partner of TLR4 in LPS recognition while the function of MD-2 multimer remains unclear. The present grant proposal will test the hypothesis that MD-2 multimer can act as an LPS decoy. In order to demonstrate this property we will have to prove that multimeric MD-2, despite its inability to bind to TLR4 and confer LPS responsiveness, is still able to bind LPS. Specifically we will: 1. Test MD-2 multimer's ability to directly bind LPS using biochemical and biophysical methods. We will also use site-directed mutagenesis to identify the region of MD-2 that is responsible for LPS recognition 2. Analyze whether addition of purified MD-2 multimer to cultures of human dendritic cells, endothelial cells, and TLR4/MD-2 expressing cell lines inhibits their responses to LPS. 3. Study the regulation of MD-2's expression and multimer formation by pro-inflammatory and antiinflammatory stimuli. These studies will augment our understanding of the molecular aspects of LPS recognition and cell activation and will characterize mechanisms to inhibit LPS action that might be exploited for therapeutic intervention.