Identifying binding partners, biological substrates and antisense oligonucleotides regulating expression of short and long ACE2.
Lead Research Organisation:
University of Southampton
Department Name: Human Development and Health
Abstract
ACE2 is the main viral entry point for SARS-CoV-2. We and others have recently demonstrated that two forms of ACE2, short and long, are expressed in airway epithelial cells, and that expression of these is under the control of independent promoters, with short ACE2 being strongly induced by IFN. Both are upregulated in response to rhinovirus infection but not SARS-CoV-2 infection.
Short ACE2 lacks the high affinity binding residues for SARS-CoV-2 spike binding, suggesting that it is not capable of SARS-CoV-2 binding. Preliminary work suggests that short ACE2 is less stable than long ACE2. Short ACE2 has a transmembrane domain but no signal peptide, and it remains unclear whether short ACE2 is located in the membrane and the mechanism of transport of ACE2. As a recently discovered molecule, little is understood about the physiological function of short ACE2 and its role in SARS-CoV-2 infectivity. In this project we aim to identify the binding partners and biological substrates of short and long ACE2 and investigate whether modulation of expression of short and long ACE2 with antisense olignucleotides can modify SARS-CoV-2 infectivity in cell models of respiratory epithelium.
Short ACE2 lacks the high affinity binding residues for SARS-CoV-2 spike binding, suggesting that it is not capable of SARS-CoV-2 binding. Preliminary work suggests that short ACE2 is less stable than long ACE2. Short ACE2 has a transmembrane domain but no signal peptide, and it remains unclear whether short ACE2 is located in the membrane and the mechanism of transport of ACE2. As a recently discovered molecule, little is understood about the physiological function of short ACE2 and its role in SARS-CoV-2 infectivity. In this project we aim to identify the binding partners and biological substrates of short and long ACE2 and investigate whether modulation of expression of short and long ACE2 with antisense olignucleotides can modify SARS-CoV-2 infectivity in cell models of respiratory epithelium.
| Description | To identify binding partners of short ACE2, we transiently expressed short and long ACE2 in respiratory epithelial cells, performed a pull-down and used mass spectrometry to identify co-precipitating proteins. While the expression of long ACE2 resulted in good yields, expression of short ACE2 was low and did not allow detection of any short ACE2 specific peptides. As this raised questions about the specificity of co-precipitated proteins, we explored an alternative approach. We commissioned the generation of polyclonal short ACE2 specific antibodies raised in rabbit and rat. Further work is needed to validate the antibodies specificity. In order to identify biological substrates of short ACE2, we established methods to detect the activity of ACE2 using a fluorescence as well as a mass spectrometric assay. Due to the unexpected difficulties of expressing reasonable amounts of recombinant short ACE2, we have used an alternative approach to express long and short ACE2 by in vitro translation. For this have we established a collaboration with Nuclera Nucleics Ltd (Cambridge, UK) and have used their novel microfluidics-based cell-free expression system to successfully express long and short ACE2. We confirmed carboxypeptidase activity for long ACE2, but were unable to detect any activity for short ACE2. However, we cannot exclude that the lack of activity is linked to the cell-free expression of short ACE2 or the used substrates. Additional work is required to confirm that short ACE2 has not a carboxypeptidase function. To modify the expression of short ACE2 we have developed a variety of oligonucleotides that interfere with ACE2 expression. We performed experiments to analyse their effect on short and long ACE2 mRNA expression, however, results were inconclusive. We therefore generated short ACE2 Crispr/Cas9 knockout cell lines and performed RNAseq analysis on these clones. Further work is needed to validate the results. |
| Exploitation Route | As a proof-of-principle we used innovative new technology provided by Nuclera Nucleics Ltd to validate our antibodies which can be adapted by others to demonstrate specificity of an antibody. Further human studies should verify stratified ACE2 type expression (by tissue type) - especially with regard to host-viral response studies. |
| Sectors | Pharmaceuticals and Medical Biotechnology Other |
| Description | We identified new ethos to avoid silo working by pooling resource and expertise. This has been described in a online blog: Discovering a new ACE2 isoform; rallying to understand SARS-CoV-2 infection | Nature Portfolio Microbiology Community January 2021 |
| First Year Of Impact | 2021 |
| Sector | Other |
| Impact Types | Cultural |
| Title | shortACE2 ABs |
| Description | We raised affinity purified polyclonal antibodies against a specific sequence in rabbit and rat. |
| Type Of Material | Antibody |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | These antibodies will allow us to identify the presents of a specific protein. |
| Description | shortACE2 monoclonal antibody |
| Organisation | National Institutes of Health (NIH) |
| Department | National Cancer Institute (NCI) |
| Country | United States |
| Sector | Public |
| PI Contribution | We contribute samples and perform analysis in this collaboration. |
| Collaborator Contribution | Our collaborator contributed a monoclonal custom made antibody that is not commercially available. |
| Impact | No outputs at this stage yet. |
| Start Year | 2021 |
| Description | Cilia Network |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | Researchers and practitioners in the Cilia Network attended a workshop where a team member gave a presentation that raised awareness and discussion around the presented data. |
| Year(s) Of Engagement Activity | 2021 |
| Description | ERS Lung Science Conference 2022 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | A team member presented our data as a poster at a conference attended by other researchers presenting, which initiated discussions afterwards and raised awareness of our research. The team member won an award for the presentation. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://openres.ersjournals.com/content/8/suppl_8/225 |
| Description | ERS conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | A team member gave a talk at a conference attended by other researchers presenting our data, which initiated discussions afterwards and raised awareness of our research. |
| Year(s) Of Engagement Activity | 2021 |
| URL | https://erj.ersjournals.com/content/58/suppl_65/OA4306 |