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首页> 美国卫生研究院文献>Cells >Host-Cell Type Dependent Features of Recombinant Human Aquaporin-4 Orthogonal Arrays of Particles—New Insights for Structural and Functional Studies
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Host-Cell Type Dependent Features of Recombinant Human Aquaporin-4 Orthogonal Arrays of Particles—New Insights for Structural and Functional Studies

机译：重组人类水通道蛋白4正交阵列的粒子的宿主细胞类型依赖性特征—结构和功能研究的新见解

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The CNS plasma-membrane water channel aquaporin-4 (AQP4) is expressed as two major isoforms able to aggregate into supramolecular assemblies known as ‘orthogonal arrays of particles’ (OAPs). OAP subnanometric features are largely unknown mainly because a method for the expression, isolation, and crystallization of integral human OAPs has not been developed. Here, the human OAP-forming isoform M23-AQP4 was expressed in insect and mammalian cell lines and AQP4 and OAP features evaluated. Native size exclusion chromatography was employed to isolate and analyze authentically folded OAPs, and neuromyelitis optica (NMO)-specific sandwich ELISA was developed to test OAP-integrity. The results demonstrate that in insect cells most AQP4 remains intracellular and unfolded and that OAPs are largely disassembled after the detergent extraction step. In mammalian cells, AQP4 showed regular plasma membrane targeting and OAPs exhibited strong post-extraction stability. Starting from the mammalian cell expression system, we isolated authentically folded OAPs. Together these data suggest a new strategy for expressing and isolating integral recombinant human OAPs and providing new insights into the cell-type dependent OAP-assembly and post-extraction stability, potentially useful to design new approaches for structural and functional studies of OAP and for other plasma membrane proteins organized into supramolecular structures.
机译：中枢神经系统血浆膜水通道水通道蛋白4（AQP4）表示为两种主要的亚型，能够聚集为称为“粒子的正交阵列”（OAPs）的超分子组装体。 OAP亚纳米特征在很大程度上是未知的，这主要是因为尚未开发出表达，分离和结晶完整人类OAP的方法。在这里，人类OAP形成同工型M23-AQP4在昆虫和哺乳动物细胞系中表达，并评估了AQP4和OAP特征。天然尺寸排阻色谱法用于分离和分析真实折叠的OAP，开发了视神经脊髓炎（NMO）特异性夹心ELISA以测试OAP完整性。结果表明，在昆虫细胞中，大多数AQP4仍保留在细胞内且未折叠，并且在去污剂提取步骤后OAP大量分解。在哺乳动物细胞中，AQP4表现出规则的质膜靶向性，而OAP表现出较强的提取后稳定性。从哺乳动物细胞表达系统开始，我们分离出了真正折叠的OAP。这些数据共同为表达和分离完整的重组人OAP提供了新的策略，并为依赖细胞类型的OAP组装和提取后的稳定性提供了新的见识，可能对设计OAP的结构和功能研究以及其他新方法很有用质膜蛋白组织成超分子结构。

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