Multimeric MD-2 as an LPS decoy

描述（由申请人提供）：作为LPS诱饵的多聚体MD-2革兰氏阴性菌脂多糖（LPS）的识别由Toll样受体4（TLR 4）、MD-2和CD 14构成的细胞表面受体复合物介导。MD-2是与TLR 4的细胞外部分相互作用并且能够直接结合LPS的分泌蛋白。MD-2以单体形式和作为二硫键连接的多聚体存在。我们发现只有MD-2单体与TLR 4结合。此外，只有MD-2单体赋予LPS对表达TLR 4的细胞系的应答性。因此，单体MD-2是TLR 4在LPS识别中的功能伴侣，而MD-2多聚体的功能尚不清楚。目前的资助提案将测试MD-2多聚体可以作为LPS诱饵的假设。为了证明这种性质，我们必须证明多聚体MD-2尽管不能结合TLR 4并赋予LPS应答性，但仍然能够结合LPS。具体来说，我们将：1。使用生物化学和生物物理方法测试MD-2多聚体直接结合LPS的能力。我们还将使用定点诱变来鉴定负责LPS识别的MD-2区域2。分析将纯化的MD-2多聚体加入人树突细胞、内皮细胞和表达TLR 4/MD-2的细胞系的培养物中是否抑制它们对LPS的应答。3.研究促炎性和炎性刺激对MD-2表达和多聚体形成的调控。这些研究将增强我们对LPS识别和细胞活化的分子方面的理解，并将表征可能用于治疗干预的抑制LPS作用的机制。

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.