A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81.
A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81.
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DOI：
10.12688/wellcomeopenres.12058.1
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发表时间：
2017
影响因子：
--
通讯作者：
McKeating JA
中科院分区：
文献类型：
--
作者：
Grove J;Hu K;Farquhar MJ;Goodall M;Walker L;Jamshad M;Drummer HE;Bill RM;Balfe P;McKeating JA
背景资料：四跨膜蛋白是存在于所有高等真核生物中的小的跨膜蛋白，其通过与伴侣蛋白的相互作用来划分细胞膜。CD 81是一种典型的四跨膜蛋白，参与多种生理和病理过程，包括作为丙型肝炎病毒（HCV）的关键进入受体。四跨膜蛋白的抗体结合可以诱导多种效应，包括肌动蛋白细胞骨架重排、MAPK-ERK信号传导的激活和细胞迁移。然而，大多数抗四跨膜蛋白抗体的表位特异性是未知的，限制了这些研究的机制解释。 研究方法：我们产生了一组特异性针对CD 81第二胞外结构域（EC2）的单克隆抗体（mAb），并用一组CD 81突变体进行了详细的表位作图。筛选所有mAb抑制HCV感染和E2-CD 81结合的能力。扫描电镜观察细胞表面CD 81的纳米级分布。 结果：抗体被分类为靶向EC2相对侧的两个表位组。我们观察到广泛的抗HCV效力是独立的表位分组，但与他们的相对亲和力细胞表面表达的CD 81。扫描电子显微镜确定了至少两个群体的CD 81，单分散和高阶组件，与四跨膜蛋白富集的微区一致。 结论：这些新的抗体提供了良好的表征工具来研究CD 81功能，包括HCV进入，并有可能提供一般四跨膜蛋白生物学的见解。
Background: Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies. Methods: We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy. Results: The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains. Conclusions: These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general.