Identification of a Heme Activation Site on the MD-2/TLR4 Complex
Identification of a Heme Activation Site on the MD-2/TLR4 Complex
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DOI：
10.3389/fimmu.2020.01370
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发表时间：
2020-06-30
影响因子：
7.3
通讯作者：
Vercellotti, Gregory M.
中科院分区：
文献类型：
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作者：
Belcher, John D.;Zhang, Ping;Vercellotti, Gregory M.
髓系分化因子-2(MD-2)与脂多糖(LPS)结合，启动Toll样受体-4(Toll-like Receptor-4，TLR4)促炎症信号转导。血红素也激活TLR4信号，但是否与MD-2相互作用尚不清楚。因此，我们检查了MD-2的一个潜在的血红素激活位点。血红素-琼脂糖凝胶和生物素-血红素/链霉亲和素琼脂糖凝胶破坏了重组MD-2，而过量的游离血红素对MD-2有抑制作用。UV/可见光光谱证实了MD-2-血红素的结合。将MD-2、TLR4、CD14和核因子-kappa B荧光素酶报告基因导入HEK293细胞，然后用亚铁血红素或脂多糖刺激HEK293细胞，以确定是否需要MD-2才能介导TLR4信号转导。亚铁血红素或脂多糖处理可激发出较强的报道活性。缺乏MD-2、TLR4或CD14质粒可阻断NF-kappa B对血红素或LPS的报告反应。在电子分析中，发现MD-2上靠近保守氨基酸W23/S33/Y34和Y36/C37/I44的两个潜在的血红素对接位点。在转染MD-2突变体W23A和Y34A的HEK293细胞中，与WT-MD-2相比，血红素诱导的NF-kappaB活性分别降低了39%和78%。同样的突变体不影响内毒素对核因子-kappaB的激活。生物素-血红素/链霉亲和素-琼脂糖凝胶对W23A和W23A/S33A/Y34A突变株MD-2的抑制作用分别比WT-MD-2低68%和80%。相反，在Y36/C37/I44 MD-2位点，突变株Y36A(相当于WT-MD-2的191%)显著增强了血红素诱导的核因子-kappaB的活性，而突变体C37A和I44A(分别为WT-MD-2的95%和92%)对其活性没有影响。综上所述，这些数据表明，血红素结合并激活MD-2上氨基酸W23和Y34上的TLR4信号。
Myeloid differentiation factor-2 (MD-2) binds lipopolysaccharide (LPS) and initiates toll-like receptor-4 (TLR4) pro-inflammatory signaling. Heme also activates TLR4 signaling, but it is unknown if heme interacts with MD-2. Therefore, we examined MD-2 for a potential heme activation site. Heme-agarose and biotin-heme/streptavidin-agarose pulled down recombinant MD-2, which was inhibited by excess free heme. UV/visible spectroscopy confirmed MD-2-heme binding. To determine whether MD-2 was required for heme-mediated TLR4 signaling, HEK293 cells were transfected with MD-2, TLR4, CD14, and an NF-kappa B luciferase reporter, and then stimulated with heme or LPS. Heme or LPS treatment elicited robust reporter activity. Absence of MD-2, TLR4 or CD14 plasmid abolished NF-kappa B reporter responses to heme or LPS.In silicoanalysis identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44. Heme-induced NF-kappa B activity was reduced by 39 and 78% in HEK293 cells transfected with MD-2 mutants W23A and Y34A, respectively, compared to WT-MD-2. NF-kappa B activation by LPS was not affected by the same mutants. Biotinyl-heme/streptavidin-agarose pulled down 68% less W23A and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the Y36/C37/I44 MD-2 site, heme-induced NF-kappa B activity was significantly increased by mutants Y36A (191% of WT-MD-2) and unchanged by mutants C37A and I44A (95 and 92%, respectively, of WT-MD-2). In conclusion, these data suggest that heme binds and activates TLR4 signaling at amino acids W23 and Y34 on MD-2.