The glycocalyx—a plethora of sugars forming a dense layer that covers the cell membrane—is commonly found on the epithelial surface of lumen forming tissue. New glycocalyx specific properties have been defined for various organs in the last decade. However, in the lung alveolar epithelium, its structure and functions remain almost completely unexplored. This is partly due to the lack of physiologically relevant, cost effective in vitro models. As the glycocalyx is an essential but neglected part of the alveolar epithelial barrier, understanding its properties holds the promise to enhance the pulmonary administration of drugs and delivery of nanoparticles. Here, using air-liquid-interface (ALI) cell culture, we focus on combining metabolic glycoengineering with glycan specific electron and confocal microscopy to visualize the glycocalyx of a recently immortalized human alveolar epithelial cell line (hAELVi). For this purpose, we applied different bioorthogonal labeling approaches to visualize sialic acid—an amino sugar that provides negative charge to the lung epithelial glycocalyx—using both fluorescence and gold-nanoparticle labeling. Further, we compared mild chemical fixing/freeze substitution and standard cytochemical electron microscopy embedding protocols for their capacity of contrasting the glycocalyx. In our study, we established hAELVi cells as a convenient model for investigating human alveolar epithelial glycocalyx. Transmission electron microscopy revealed hAELVi cells to develop ultrastructural features reminiscent of alveolar epithelial type II cells (ATII). Further, we visualized extracellular uni- and multilamellar membranous structures in direct proximity to the glycocalyx at ultrastructural level, indicating putative interactions. The lamellar membranes were able to form structures of higher organization, and we report sialic acid to be present within those. In conclusion, combining metabolite specific glycoengineering with ultrastructural localization presents an innovative method with high potential to depict the molecular distribution of individual components of the alveolar epithelial glycocalyx and its interaction partners.

中文翻译：

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代谢糖工程使肺泡上皮细胞系 hAELVi 糖萼中唾液酸的超微结构可视化成为可能

糖萼——形成覆盖细胞膜的致密层的过多糖——常见于管腔形成组织的上皮表面。在过去的十年中，已经为各种器官定义了新的糖萼特定特性。然而，在肺泡上皮中，其结构和功能几乎完全未被探索。这部分是由于缺乏生理相关的、具有成本效益的体外模型。由于糖萼是肺泡上皮屏障的重要但被忽视的部分，了解其特性有望增强药物的肺部给药和纳米颗粒的递送。在这里，使用气液界面 (ALI) 细胞培养，我们专注于将代谢糖工程与聚糖特异性电子和共聚焦显微镜相结合，以可视化最近永生化的人肺泡上皮细胞系 (hAELVi) 的糖萼。为此，我们应用了不同的生物正交标记方法来可视化唾液酸（一种为肺上皮糖萼提供负电荷的氨基糖），同时使用荧光和金纳米颗粒标记。此外，我们比较了温和的化学固定/冷冻替代和标准细胞化学电子显微镜嵌入协议的对比糖萼的能力。在我们的研究中，我们建立了 hAELVi 细胞作为研究人肺泡上皮糖萼的便捷模型。透射电子显微镜显示 hAELVi 细胞发展出让人联想到肺泡上皮 II 型细胞 (ATII) 的超微结构特征。此外，我们在超微结构水平上观察到直接靠近糖萼的细胞外单层和多层膜结构，表明假定的相互作用。层状膜能够形成更高组织的结构，我们报告唾液酸存在于其中。总之，将代谢物特异性糖工程与超微结构定位相结合，提供了一种具有高潜力的创新方法，可以描述肺泡上皮糖萼及其相互作用伙伴的各个成分的分子分布。我们在超微结构水平上观察到直接靠近糖萼的细胞外单层和多层膜结构，表明假定的相互作用。层状膜能够形成更高组织的结构，我们报告唾液酸存在于其中。总之，将代谢物特异性糖工程与超微结构定位相结合，提供了一种具有高潜力的创新方法，可以描述肺泡上皮糖萼及其相互作用伙伴的各个成分的分子分布。我们在超微结构水平上观察到直接靠近糖萼的细胞外单层和多层膜结构，表明假定的相互作用。层状膜能够形成更高组织的结构，我们报告唾液酸存在于其中。总之，将代谢物特异性糖工程与超微结构定位相结合提供了一种创新方法，具有描述肺泡上皮糖萼及其相互作用伙伴的各个组分的分子分布的潜力。