Fighting COVID -19: Spotlight on cell-based assay

As the COVID-19 pandemic is getting severe, never before have so many of the world’s researchers focused urgently on a single topic. Our company, ACROBiosystems has also contributed a lot of efforts against COVID 19.

The spike of SARS-CoV-2 is a highly glycosylated homologous trimer composed of two subunits, S1 and S2. ^[1-2] Same as SARS-CoV-1, the spike protein of SARS-CoV-2 facilitates viral entry into the target cell exploiting the Angiotensin-converting enzyme 2 (ACE2) receptor. ^[1-3] A serine protease, TMPRSS2, is also involved in this process, responsible for priming of the spike protein. The proteolytic cleavage results in the shedding of the S1 subunit and ACE2. ^[3,4] Due to the shedding, you will see a much weaker signal under flow cytometry analysis when investigating the spike protein or S1 protein binding with the ACE2 on the cell surface. This is different from the strong signal you get from protein-based ELISA or SPR analysis.

ACROBiosystems has successfully developed a flow cytometry assay to investigate the binding between S1 protein/ S1 RBD protein and ACE2, thus can be used for neutralizing antibody screening.

The transmembrane protease ADAM17 is also reported to be involved in the process of infecting host cells ^[5,6]. The binding of S protein to ACE2 activates ADAM17, which catalyzes the ACE2 shedding and helps with the virus entry into the cell ^[6-9]. Back to 2010, Haga et al. demonstrated that SARS-CoV-1 spike protein can induce ACE2 shedding in the VERO E6 cell model, while TAPI-2, the inhibitor of ADAM17, can prevent ACE2 shedding ^[6,9]. ACROBiosystems repeated the same assay using SARS-CoV-2 spike protein. A fluorescent peptide substrate was used to detect the ACE2 activity in cell culture supernatant. We confirmed that the S1 protein of SARS-CoV-2 could induce the shedding of ACE2 in VERO E6 cells. (Figure below)

SARS-CoV-2 (COVID-19) S1 protein, Mouse IgG2a Fc Tag

Cat.No.：S1N-C5257

SARS-CoV-2 (COVID-19) S1 protein, His Tag

Cat.No.：S1N-C52H3

Related Product:

S1 Protein His Tag（S1N-C52H3）

S1 Protein mFc Tag（S1N-C5257）

Reference:

1. Structural basis for therecognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444–1448(2020).

2. Cryo-EM Structure of the2019-nCoV Spike in the Prefusion Conformation. Science 367, 1260-1263(2020).

3. SARS-CoV-2 Cell entry dependson ACE2 and TMPRSS2 and Is blocked by a clinically proven protease Inhibitor.Cell 181, 1–10(2020).

4. Structural Basis of SARS-CoV-2Spike Protein Priming by TMPRSS2. https://doi.org/10.1101/2020.04.21.052639.

5. Tumor necrosis Factor-a convertase(ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratorysyndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2(ACE2). JBC 280,30113–30119(2005).

6. Modulation of TNF-a-convertingenzyme by the spike protein of SARS-CoV and ACE2 induces TNF-a production andfacilitates viral entry. PNAS 105, 7809–7814(2008)

7. Trilogy of ACE2: A peptidase inthe renin–angiotensin system, a SARS receptor, and a partner for amino acidtransporters. Pharmacology & Therapeutics 128, 119–128(2010).

8. TACE antagonists blocking ACE2shedding caused by the spike protein of SARS-CoV are candidate antiviralcompounds. Antiviral Research, 85, 551–555(2010).

9. Differential Downregulation of ACE2 by the SpikeProteins of Severe Acute Respiratory Syndrome Coronavirus and Human CoronavirusNL63. VIROLOGY, 84, 1198–1205(2010).