The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
Biomaterials. 2010 Mar;31(7):1935-43. doi: 10.1016/j.biomaterials.2009.10.015. Epub 2009 Oct 23.
The cytokine lymphotoxin-alpha (LT alpha) activates various biological functions through its three receptor subtypes, tumor necrosis factor receptor 1 (TNFR1), TNFR2 and herpes virus entry mediator (HVEM), but the relative contribution of each receptor to each function is unclear. Therefore it is important to create mutant LT alpha with receptor selectivity for optimized cancer therapy and the analysis of receptor function. Here, we attempted to create a lysine-deficient mutant LT alpha with TNFR1-selective bioactivity using a phage display technique. We obtained the TNFR1-selective mutant LT alpha R1selLT, which contained the mutations K19N, K28Q, K39S, K84Q, K89V, and K119H. Compared with wild-type LT alpha (wtLT alpha), R1selLT showed several-fold higher bioactivity via TNFR1 but 40-fold lower bioactivity via TNFR2. Kinetic association-dissociation parameters of R1selLT with TNFR2 were higher than those of wtLT alpha, whereas these parameters of R1selLT with TNFR1 were lower than those of wtLT alpha, suggesting that destabilization of the R1selLT-TNFR2 complex causes the decreased bioactivity of R1selLT on TNFR2. We also showed that the K84Q mutation contributed to the enhanced activity via TNFR1, and K39S lowered activity via TNFR2. R1selLT likely will be useful in cancer therapy and in analysis of the LT alpha structure-function relationship.
细胞因子淋巴毒素-α（LTα）通过其三种受体亚型，肿瘤坏死因子受体 1（TNFR1）、TNFR2 和疱疹病毒进入介体（HVEM），激活各种生物学功能，但每种受体对每种功能的相对贡献尚不清楚。因此，创建具有受体选择性的突变型 LTα对于优化癌症治疗和受体功能分析非常重要。在这里，我们尝试使用噬菌体展示技术创建具有 TNFR1 选择性生物活性的赖氨酸缺陷型 LTα突变体。我们获得了 TNFR1 选择性突变体 LTα R1selLT，其包含突变 K19N、K28Q、K39S、K84Q、K89V 和 K119H。与野生型 LTα（wtLTα）相比，R1selLT 通过 TNFR1 显示出几倍更高的生物活性，但通过 TNFR2 显示出 40 倍更低的生物活性。R1selLT 与 TNFR2 的动力学关联-解离参数高于 wtLTα，而 R1selLT 与 TNFR1 的这些参数低于 wtLTα，表明 R1selLT-TNFR2 复合物的不稳定性导致 R1selLT 对 TNFR2 的生物活性降低。我们还表明，K84Q 突变有助于通过 TNFR1 增强活性，而 K39S 通过 TNFR2 降低活性。R1selLT 可能在癌症治疗和 LTα 结构-功能关系分析中有用。
