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Technologies for Emerging Infectious Diseases

Technologies for Emerging Infectious Diseases - Poster Session A

Poster K13 - Cathepsin proteolytic cleavage of SARS-CoV-2 spike protein and identification of cleavage sites

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Proteases are implicated in the infection process of SARS-CoV-2, the virus causing the COVID-19 pandemic. At the cell surface, furin and TMPRSS2 proteases can cleave the spike protein into S1 and S2 subunits, leading to the virion fusing to and entering the membrane of the target cell. After endocytosis, proteases (including cathepsins B and L) facilitate endolysosomal escape in acidic environments. While spike protein activation is primarily facilitated by these proteases, the identification of other proteases involved in SARS-Cov-2 entry into cells is important for the development of new therapeutic targets. Cathepsins K, S, and V all share 60-80% sequence homology with cathepsin L, implicating them as potentially being able to cleave the SARS-CoV-2 spike protein. In patients with preexisting conditions such as cardiovascular disease, sickle cell anemia, emphysema, and diabetes, all of which are associated with an upregulation in these cathepsins, there is an elevated risk of severe COVID-19 and mortality. This suggests that cathepsins K, S, and V may cleave the SARS-CoV-2 spike protein. Understanding cathepsin cleavage of the SARS-CoV-2 virus may provide new therapeutic targets for the treatment or prevention of SARS-CoV-2 infection.

Materials and

Methods:  Recombinant proteins were produced at NCI’s Protein Expression Laboratory. SARS-CoV-2 spike proteins were expressed in human HEK293T cells, cathepsin S in Tni-FNL baculovirus, and cathepsin K in P pastoris yeast. Cathepsins and spike proteins were verified by SDS-PAGE and Western Blotting. To determine proteolytic activity of these recombinant cathepsins, DQ Gelatin was used as a fluorogenic substrate, incubated with increasing concentrations of cathepsins S and K, and fluorescence measured every five minutes using a BioTek Synergy plate reader for two hours. Western blotting and peptide mapping using LC/MS were employed to determine cleavage locations. Cathepsins were co-incubated with spike protein and aliquots taken from 0 to 60 minutes, prior to stopping reaction with E64 inhibition and boiling for 5 minutes. Proteins were then loaded for SDS-PAGE in identical gels where aliquots from reaction were either (1) transferred to membranes for Western blotting or (2) Coomassie-stained and bands extracted for trypsin digestion and LC/MS. For Western Blots, a multiplex assay was done with antibodies for the N-Terminal and the S2 domains to identify sizes of cleaved products. LC/MS bands were submitted to mass spectrometry and peptide mapping.

Results, Conclusions, and Discussions: Gelatinase activity of cathepsins K, L, S, and V was measured using a DQ gelatin assay to determine the lowest effective concentration of each cathepsin. All recombinant cathepsins were able to cleave gelatin; in fact, very small micromolar amounts of the cathepsins were needed to work as a potent gelatinase (Figure 1). The ideal cathepsin amount for the DQ gelatin assay with a 125 µL volume was found to be 5 picomoles, which demonstrated that the cathepsin cleavage of gelatin increased over time for all cathepsins.

Western blotting of co-incubated wild type spike protein with cathepsin K (Figure 2A) and cathepsin S (Figure 2B) demonstrated the locations of cathepsin cleavage of the spike protein, with the S2 domain highlighted in red, and the N-Terminal domain highlighted in green. These results demonstrate that cathepsins S and K cleave the SARS-CoV-2 spike protein. The disappearance of NTD and S2 signals over time suggests that the cathepsins are either cleaving at the antibody binding sites or that the fragments are too small to detect. Cathepsins K and S are both initially cleaving the NTD side of the protein into 2 pieces – an approximately 105 kDa fragment connected to the S2 domain and a 75 kDa fragment connected to the NTD (Figure 3). However, as the cleavages continue, separate cleavages are observed in the cathepsin K and cathepsin S incubations. This shows that cathepsin K and cathepsin S share some cleavage sites but also have some unique cleavage sites.

Trypsin-digested cathepsins and spike proteins were analyzed using liquid chromatography mass spectrometry in order to define what peptides were present prior to cathepsin cleavage of spike proteins. Peptides were successfully mapped from SDS-PAGE gel band excisions with 65-92% sequence coverage. The peptide sequences present in each cathepsin and spike protein sample will then be compared to samples with cathepsin and spike protein co-incubated in order to determine where the cathepsins are cleaving spike protein.

Acknowledgements (Optional):