The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

Manna, Vincent J.; Caradonna, Salvatore J.

Coronavirus Disease 2019 (COVID-19) is transmitted through respiratory droplets containing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) particles. Once inhaled, SARS-CoV-2 particles gain entry into respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). It is known that ACE2 functions within the renin-angiotensin system to regulate blood pressure, fluid homeostasis and inflammation. However, it is largely unknown what roles ACE2 has in ciliated cells of the airway. Therefore, understanding the function and nature of ACE2 within airway tissue has become an essential element in combatting the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells isolated from nasal turbinates of donors and the air-liquid interface (ALI) model of differentiation. Using ALI tissue we cloned transcripts for three distinct variants of ACE2, one of which encodes the full-length ACE2 protein, the other two transcripts are truncated isoforms that had only been predicted to exist via sequence analysis software. We demonstrate that all three isoforms have the capacity to be glycosylated, a known modification of full-length ACE2. Immunofluorescence microscopy of individual ACE2 isoform transfected cells reveals distinct localization of variant 1 relative to X1 and X2. Double staining immunohistochemistry of ALI tissue using antibodies to either the N-term or C-term region of ACE2 revealed distinct and overlapping signals in the apical cytosol of ciliated cells. Most notably only the ACE2 C-term antibody displayed plasma-membrane localization in ciliated cells. We also observed a decrease in the total amount of ACE2 in ALI tissue derived from a 33 year-old male donor when compared to a 34 year-old female donor, thus there may be variation in the abundance of ACE2 protein in the airway among the population. Together, our data begins to highlight the dynamic status of the ACE2 protein in airway mucociliary tissue and we propose multiple ACE2 parameters that may impact an individual’s susceptibility to SARS-CoV-2. These parameters include the balance of cytosolic versus membrane bound ACE2, isoform expression levels, maintenance of post-translational modifications and the impact of genetic, environmental and lifestyle factors on these processes.