Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders.
Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders.
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调节与耳聋和皮肤病有关的突变连接蛋白 26 半通道的人单克隆抗体的设计和表征。
DOI：
10.3389/fnmol.2017.00298
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发表时间：
2017
影响因子：
4.8
通讯作者：
Mammano F
中科院分区：
文献类型：
--
作者：
Xu L;Carrer A;Zonta F;Qu Z;Ma P;Li S;Ceriani F;Buratto D;Crispino G;Zorzi V;Ziraldo G;Bruno F;Nardin C;Peres C;Mazzarda F;Salvatore AM;Raspa M;Scavizzi F;Chu Y;Xie S;Yang X;Liao J;Liu X;Wang W;Wang S;Yang G;Lerner RA;Mammano F
背景资料：导致连接蛋白通道的性质、调节或表达变化的突变与28种不同的人类遗传性疾病有关。其中八种是由连接蛋白26（Cx 26）的变体引起的，连接蛋白26是一种与内耳和皮肤中的细胞-细胞信号传导密切相关的蛋白质。缺乏具有明确作用机制的无毒药物对突变连接蛋白引起的疾病的治疗干预构成严重障碍。特别地，特异性调节连接蛋白半通道功能而不影响间隙连接通道的分子被认为对于研究连接蛋白半通道在生理以及病理条件中的作用是最重要的。单克隆抗体是近三十年来发展起来的一类重要的生物治疗药物。重组方法允许从具有大多样性的文库中快速选择和改进单克隆抗体。研究方法：通过筛选在噬菌体中表达的人单链可变片段（scFv）抗体的组合文库，我们鉴定了结合Cx 26的细胞外表位的候选物。我们的特点是抗体的作用，使用各种生物化学和生物物理测定在HeLa细胞，小鼠耳蜗和人角质形成细胞的器官型培养。结果如下：我们确定该抗体是连接蛋白26形成的半通道的显著有效、无毒和完全可逆的抑制剂，并且不影响通过间隙连接通道的直接细胞-细胞通信。重要的是，我们还证明，抗体有效地抑制过度突变型Cx 26半通道牵连常染色体显性非综合征性听力障碍伴随角膜炎和hystrix样鱼鳞病耳聋（KID/HID）综合征。我们解决了抗体的晶体结构，确定了对结合至关重要的残基，并使用分子动力学来揭示其作用机制。结论：尽管需要进一步研究来验证抗体在体内的作用，但本文所述的方法可以扩展到选择针对由其他连接蛋白同种型组成的半通道的抗体，并因此靶向与过度活跃的半通道相关的其他病理。我们的研究强调了这种方法的潜力，并确定连接蛋白作为治疗靶点，通过筛选表达人随机抗体的噬菌体展示文库。
Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.
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影响因子：
5.8
作者：
Brenke, Ryan;Hall, David R.;Kozakov, Dima
通讯作者：
Kozakov, Dima
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.5
作者：
Bosen, Felicitas;Schuetz, Melanie;Willecke, Klaus
通讯作者：
Willecke, Klaus
DOI：
10.1073/pnas.1616061113
发表时间：
2016-11-15
影响因子：
11.1
作者：
Ceriani, Federico;Pozzan, Tullio;Mammano, Fabio
通讯作者：
Mammano, Fabio
影响因子：
3.7
作者：
Clair, Caroline;Combettes, Laurent;Van Nlieu, Guy Tran
通讯作者：
Van Nlieu, Guy Tran