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首页> 外文期刊>Frontiers in Molecular Neuroscience >Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders
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Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

机译：人单克隆抗体的设计和表征，可调节与耳聋和皮肤疾病有关的突变型连接蛋白26半通道

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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.
机译：背景：导致连接蛋白合成通道的性质，调控或表达发生变化的突变与28种人类遗传疾病有关。其中的八种来自连接蛋白26（Cx26）的变异体，它是内耳和皮肤细胞信号传导的关键蛋白。缺乏确定作用机制的无毒药物严重阻碍了突变连接蛋白引起的疾病的治疗干预。特别地，对于研究连接蛋白半通道在生理以及病理状况中的作用的研究，特异性地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是最重要的。在过去的三十年中开发的单克隆抗体已成为治疗生物学最重要的一类。重组方法可从多样性很大的文库中快速选择和改进单克隆抗体。方法：通过筛选在噬菌体中表达的人单链片段可变（scFv）抗体组合文库，我们鉴定了结合Cx26细胞外表位的候选基因。我们在HeLa细胞，小鼠耳蜗的有机型培养物和人角质形成细胞衍生的细胞中使用多种生化和生物物理分析表征了抗体的作用。结果：我们确定该抗体是由连接蛋白26形成的半通道的显着有效，无毒且完全可逆的抑制剂，并且不会影响通过间隙连接通道的直接细胞间通讯。重要的是，我们还证明了该抗体可有效抑制常染色体显性非综合征性听力障碍伴有角膜炎和hystrix样鱼鳞病性耳聋（KID / HID）综合征所涉及的过度突变Cx26半通道。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，并利用分子动力学揭示了其作用机理。结论：尽管有必要进行进一步的研究以验证抗体在体内的作用，但此处描述的方法可以扩展为选择针对由其他连接蛋白同工型组成的半通道的抗体，从而针对与多动半通道相关的其他病理。我们的研究突出了这种方法的潜力，并通过筛选表达人类随机抗体的噬菌体展示文库确定连接蛋白作为可治疗的靶标。

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