Comparison of lipopolysaccharide-binding functions of CD14 and MD-2
Comparison of lipopolysaccharide-binding functions of CD14 and MD-2
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DOI：
10.1128/cdli.12.11.1292-1297.2005
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发表时间：
2005-11-01
期刊：
影响因子：
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通讯作者：
Fukudome, K
中科院分区：
文献类型：
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作者：
Koraha, J;Tsuneyoshi, N;Fukudome, K
细菌外膜中的脂多糖(LPS)在被细胞表面Toll样受体4/MD-2复合体识别之前，必须经过LPS结合蛋白和CD14的处理。CD14与脂多糖结合蛋白提取的单体脂多糖形成复合体，并将其传递到细胞表面的信号复合体。在之前的研究中，我们利用细菌表达系统制备了具有功能的重组MD-2。我们在大肠杆菌中以硫氧还蛋白融合蛋白的形式表达了重组蛋白，并证明了其与内毒素的特异性结合。在本研究中，我们用同样的方法制备了重组CD14融合蛋白。含有151个氨基末端残基的重组蛋白可与内毒素特异结合。该区域包含一个亲水区和前三个富含亮氨酸的重复序列(LRR)。LRRs似乎有助于结合，因为该区域的移除导致结合功能的降低。与重组MD-2结合的内毒素对洗涤剂有抵抗力。另一方面，在低浓度的洗涤剂存在下，阻止了与CD14的结合。在人MD-2的情况下，需要LpxM添加的内毒素的二级肉豆蔻基链才能结合。缺乏肉豆蔻链的非致病性五酰内毒素突变体不与MD-2结合，但与CD14正常结合。CD14的较宽的内毒素结合谱可能允许识别多种病原体，而CD14与内毒素结合的低亲和力允许捕获的物质传递给MD-2。
Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopollysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein in Escherichia coli as a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophillic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyll LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2.