Fig. 6: dasTMPRSS2 efficiently cleaves recombinant SARS-CoV-2 Spike protein constructs at multiple sites.
From: Structure and activity of human TMPRSS2 protease implicated in SARS-CoV-2 activation
a, The trimeric Spike protein (depicted here in its monomer form) is presented at the surface of the viral membrane and binds the transmembrane human ACE2 receptor through its RBD. Three distinct TMPRSS2 protease cleavage sites are predicted (indicated by scissors). b, Schematic map of known (S1 and S2) and inferred protein fragments after TMPRSS2 treatment, as derived from protein bands (c–g) with the indicated approximate molecular weights (in parentheses) produced as a result of 0–3 protease cleavage events. Recombinant S protein is cleaved at the S1/S2 site, at an unknown site within S2 termed the X/Y cleavage site and within RBD termed the RBD–TMPRSS2 (RT) site that is spanned by a disulfide bond. Fragments containing RBD are highlighted in red, and cut patterns denoted with an asterisk are only detected using S1/S2 KO S protein, HexaPro. c, HexaFurin S protein is converted to S1 and S2 band fragments after 16 h incubation with 10 U of recombinant furin protease (NEB). Mw, molecular weight. d, dasTMPRSS2 fully converts untreated HexaFurin to S1 and S2 fragments within 5 min. e, HexaPro S protein treated with 300 nM TMPRSS2 for 30 min is cleaved at both the X/Y and RBD–TMPRSS2 cleavage sites, with the latter cleavage event apparent only under reducing (R) SDS–PAGE conditions rather than nonreducing (NR). f, Recombinant S protein constructs containing only the RBD and an RBD-Fc fusion are cleaved at the RBD–TMPRSS2 cleavage site after 120 min of TMPRSS2 incubation. g, Western blot analysis of HexaFurin S protein digestions with an RBD-specific primary antibody shows the expected S1 band fragment, the N + RBD fragment and several intermediate sized fragments also containing RBD. All gels and blots are consistent with n = 3 independent biological experiments.
