Manna Vincent J, Choi Hana, Rotoli Shawna M, Caradonna Salvatore J
Department of Molecular Biology, Graduate School of Biomedical Sciences and School of Osteopathic Medicine, Rowan University, Stratford, NJ, United States.
BBA Adv. 2022;2:100044. doi: 10.1016/j.bbadva.2022.100044. Epub 2022 Feb 12.
Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual's susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.
一旦被吸入，严重急性呼吸综合征冠状病毒2（SARS-CoV-2）颗粒通过与血管紧张素转换酶2（ACE2）相互作用进入呼吸道纤毛细胞。鉴于2019冠状病毒病大流行，了解气道组织中ACE2的性质已成为近期的一个重点。利用原代人鼻上皮细胞和气液界面（ALI）分化模型生成了气道黏液纤毛组织。利用ALI组织，鉴定出ACE2的三种不同转录变体。一种转录本编码已记录的全长ACE2蛋白。另外两种转录本是独特的截短异构体，直到最近才通过序列分析软件预测其存在。定量PCR显示，在气道黏液纤毛上皮细胞分化过程中，所有三种转录变体均有表达。对在细胞系中异位表达的单个ACE2蛋白异构体进行免疫荧光分析，发现它们在质膜定位和与SARS-CoV-2刺突受体结合域相互作用方面具有相似的能力。使用针对ACE2 N端或C端的抗体对分化的ALI组织进行免疫组织化学分析，发现细胞中的信号既有重叠又有差异，最显著的是只有ACE2 C端抗体显示出质膜定位。我们还证明，与ACE2转染的细胞系相比，ALI组织中ACE2蛋白的脱落情况不同，并且ACE2从ALI组织的顶端和基底表面释放。总之，我们的数据突出了气道黏液纤毛组织中ACE2转录本和蛋白的各个方面，这些方面可能代表影响个体对SARS-CoV-2感染易感性或2019冠状病毒病严重程度的变量。
