School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China.
Protein J. 2011 Apr;30(4):281-9. doi: 10.1007/s10930-011-9330-4.
A domain at the NH(2) terminal (N-terminal) of tumor necrosis factor receptor (TNFR) termed the pre-ligand binding assembly domain (PLAD). The finding that PLAD can mediate a selective TNFR assembly in previously researches provides a novel target to the prevention of TNFR signaling in immune-mediated inflammatory diseases (IMID). In this study, a natural N-terminal TNFR1 PLAD was obtained for the first time through the methods of GST-tag fusion protein expression and enterokinase cleavage. After purification with a Q Sepharose Fast Flow column, a natural N-terminal TNFR1 PLAD which purity was up to 95%, was obtained and was identified using Nano LC-ECI-MS/MS. Secondary structure analysis of PLAD was carried out using circular dichroism spectra (CD). After that, the TNFR1 PLAD in vitro anti-TNFα activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNFα bioactivity mainly through TNFR1. It infers an effective and safe strategy for treating variety of IMID with a low risk of side effects in future.
肿瘤坏死因子受体（TNFR）NH2 末端（N 端）的一个结构域，称为前配体结合组装域（PLAD）。先前的研究发现，PLAD 可以介导 TNFR 的选择性组装，为预防免疫介导的炎症性疾病（IMID）中的 TNFR 信号提供了一个新的靶点。在这项研究中，我们首次通过 GST 标签融合蛋白表达和肠激酶切割的方法获得了天然的 N 端 TNFR1 PLAD。通过 Q Sepharose Fast Flow 柱纯化后，获得了纯度高达 95%的天然 N 端 TNFR1 PLAD，并通过纳升液相色谱-电喷雾串联质谱（Nano LC-ECI-MS/MS）进行鉴定。使用圆二色光谱（CD）进行 PLAD 的二级结构分析。然后，测定了 TNFR1 PLAD 的体外抗 TNFα 活性和 TNFR1 的特异性亲和力。结果表明，天然的 N 端 TNFR1 PLAD 可以选择性地抑制 TNFα 的生物活性，主要通过 TNFR1。这为未来治疗多种 IMID 提供了一种有效且安全的策略，副作用风险低。
