Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2, 20126 Milano, Italy.
Biochemistry. 2009 Dec 29;48(51):12337-44. doi: 10.1021/bi901601b.
Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin [lipopolysaccharide (LPS)] to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP.CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide ([(3)H]LOS) from Neisseria meninigitidis to MD-2.TLR4, resulting in a reduced level of formation of a ([(3)H]LOS.MD-2.TLR4(ECD))(2) (M(r) approximately 190000) complex. This effect was due to inhibition of the transfer of [(3)H]LOS from aggregates in solution to sCD14 with little or no effect on [(3)H]LOS shuttling from [(3)H]LOS.sCD14 to MD-2. These compounds also inhibited transfer of the [(3)H]LOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of [(3)H]LOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2.TLR4.
具有正电荷氨基或铵盐和两个亲脂链的天然糖衍生的合成分子已被证明可在体外和体内抑制 TLR4 的激活。为了研究这类分子的作用机制，我们研究了它们与结合和转运内毒素[脂多糖（LPS）]至 TLR4 的细胞外成分的可能相互作用，即 LBP、CD14 和 MD-2。抑制 TLR4 激活的分子抑制了源自脑膜炎奈瑟菌三氚标记脂寡糖[（3）H]LOS 聚集体的内毒素单体与 MD-2.TLR4 的 LBP.CD14 依赖性转移，导致形成[（3）H]LOS.MD-2.TLR4(ECD)）（2）（M(r) 约 190000）复合物的水平降低。这种效应是由于抑制了溶液中聚集体的[（3）H]LOS 从 sCD14 的转移，而对[（3）H]LOS 从[（3）H]LOS.sCD14 向 MD-2 的转移几乎没有影响。这些化合物还抑制了全长 CD14 向截短的、多组氨酸标记的 CD14 转移[（3）H]LOS 单体。三种不同的抑制 TLR4 激活的合成化合物观察到[（3）H]LOS 在两种形式的 CD14 之间转移的剂量依赖性抑制，但另一种缺乏 TLR4 拮抗活性的结构相关类似物则没有。饱和转移差异（STD）NMR 数据表明，合成 TLR4 拮抗剂通过其亲脂链主要通过直接与 CD14 结合来介导合成化合物对 CD14 的结合。综上所述，我们的研究结果强烈表明，这些化合物通过竞争性占据 CD14 来抑制内毒素诱导的 TLR4 激活，从而降低激活内毒素向 MD-2.TLR4 的传递水平。
