Roles of glycosylation and redox states on SARS-CoV-2 spike protein actions

Abstract The pandemic of Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The viral fusion protein, spike protein, binds to its receptor Angiotensin-Converting Enzyme 2 (ACE2) to enter host cells. Further, the S1 subunit of the spike protein may be cleaved off the virus and affect various organs. In addition to tightly binding to ACE2, S1 spike protein also enhances the peptidase activity of ACE2 and activate cell signaling. The present study compared effects of recombinant S1 spike proteins expressed in HEK cells and in E. coli. The spike protein expressed in HEK cells is heavily glycosylated and binds strongly to ACE2, while the protein expressed in E. coli is not glycosylated and exhibits no ACE2 binding. The SulfoBiotics Protein Redox State Monitoring assay determined that cysteine residues of the spike protein expressed in HEK cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of the protein expressed in HEK cells by Glycopeptidase F attenuates the ACE2 binding, although not completely, and the oxidation of the E. coli protein by hydrogen peroxide enhances the binding. In contrast to the highly distinct ACE2 binding capacities of spike proteins expressed in HEK cells and in E coli, spike proteins expressed in both systems enhance the ACE2 peptidase activity and promote cell signaling. These results suggest that the glycosylated state contributes to the binding of the spike protein to ACE2 and introduce a new concept of the regulation of the spike protein-ACE2 binding by redox mechanisms. Glycosylation and redox states, however, do not define the abilities of the spike protein to enhance the ACE2 enzyme activity or promote cell signaling. Supported by grants from NIH (R03 AG71596, R21 AG73919)