Discovering a new ACE2 isoform; rallying to understand SARS-CoV-2 infection

COVID-19 forced a suspension to our primary ciliary dyskinesia (PCD) diagnostic service. Re-purposing nasal epithelial cell RNAseq data and bioresource, three University of Southampton groups joined forces and found a new “short” isoform of ACE2, the cell surface receptor for SARS-CoV-2 virus.

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In March 2020 the Southampton PCD group saw the last patients before lockdown, and we asked ourselves what we could do to support COVID-19 pandemic research.  Given our expertise in ciliated nasal cell culture models, our bioresource of human nasal cells, existing RNAseq data for nasal epithelium and collaborators in functional genomics and virology, we could quickly repurpose our lab to investigate SARS-CoV-2 entry, focussing on ACE2.  The Southampton Functional Genomics Group rapidly identified what we believed to be a novel transcript of ACE2, predicted to encode a smaller ACE2 protein, which we named short ACE2.

Along the corridor, the Brooke laboratory was already investigating ACE2 expression in the lower airway. They were particularly interested in interferon (IFN) regulation of ACE2 as an antiviral defence pathway, particularly in response to rhinovirus, and its role in exacerbations of severe asthma.  Western blots of bronchial epithelial cell lysates revealed a ~50 kDa anti-ACE2 band as well as the expected ACE2 100 kDa band. This hinted at a degradation product, or tantalisingly, a novel isoform of ACE2. 

The PCD Lab, Functional Genomics Group and Brooke Lab, quickly joined forces to investigate this novel ACE2 isoform, meeting regularly online to plan experiments and discuss results. As we progressed, we were joined by interdisciplinary researchers from across University of Southampton, particularly from the NIHR Southampton Biomedical Research Centre and the Institute for Life Sciences. We were supported by a £10,000 grant from the local AAIR Charity, and otherwise ‘begged and borrowed’ to resource the project. We knew that bulk and single-cell RNA-sequencing demonstrated low levels of ACE2 expression across a range of tissues, with higher levels in the airways; also that there was a decreasing expression gradient from the upper to the lower airways.  ACE2 was highest expressed in goblet and ciliated cells of the nasal epithelium, and was shown to localize to the ciliated membrane by immuno-fluorescence labelling. This made sense to us and it was consistent with the nose being the first entry point of respiratory viral infection.

              In our paper, which was published with two other manuscripts in Nature Genetics, we detail our discovery, then characterize the expression of short ACE2.  We show that short ACE2 is expressed in human nasal and bronchial respiratory epithelia, the main site of SARS-CoV-2 infection. Interestingly, short ACE2 does not contain the sequences that bind SARS-CoV-2 spike protein, which are required for viral entry. In airway cells, short ACE2 is up-regulated in response to IFN and to infection with rhinovirus, but not in response to SARS-CoV-2 infection. While the function of short ACE2 is still unknown, there is a possibility that short ACE2 expression plays a role during SARS-CoV-2 and other respiratory virus infections

New short ACE2 is expressed in differentiated airway epithelial cells, and is immuno-localised to cilia.  Computer modelling of short ACE2 shows it is missing most of the SARs-CoV-2 binding residues. The IFN inducible short ACE2 is upregulated in response to rhinovirus but not SARs-CoV-2 infection (see our paper).    

The discovery of short ACE2 has implications for planning and interpreting studies reporting ACE2 expression levels. Our findings should be considered when selecting reagents for future studies of ACE2 expression in tissues relevant to SARS-CoV-2 viral infection, bearing in mind the genomic region targeted by primer sets and the epitopes recognized by antibodies.        

Before the pandemic we would not have envisaged the possibility of publishing an important new discovery within nine months, supported by minimal funding and resources. We demonstrate the successful repurposing of existing resources, mobilisation of enthusiastic collaborators and miracle that can be achieved with £10,000; the catalyst was the dramatic situation of a global pandemic. UKRI have funded our ongoing research to further explore the functions of short ACE2, particularly in relation to SARS-CoV-2.

Claire Jackson

Research Fellow, PCD Scientist, University of Southampton

Claire L Jackson PhD is a Research Fellow and primary ciliary dyskinesia (PCD) scientist at the University of Southampton Faculty of Medicine and UHSFT, with expertise in ciliated nasal epithelial cell modelling, cilia imaging and PCD diagnostics and research.