Rapid Degradation of SARS-CoV-2 Spike S Protein by A Specific Serine Protease

Abstract

The S protein of SARS-CoV-2 is a crucial structural and functional component for virus entry. Due to the constant mutation of the virus, there are very limited ways to prevent and control COVID-19. This experiment used a macroscopic SDS-PAGE method and proved that the S protein of wild-type SARS-CoV-2 virus, especially the S1 subunit, is very sensitive to alkaline serine protease with acidic pI (ASPNJ), NJ represents Neanthes japonica (Izuka) from which ASP is purified). ASPNJ cleaves proteins when the carbonyl group of the peptide bond is contributed by arginine or lysine. ASPNJ can degrade the S protein very quickly and effectively in vitro with relative selectivity. It can be inferred that the S, S1 and RBD of SARS-CoV-2 variants can also be easily degraded by ASPNJ. This rapid and strong degradation of the S protein by ASPNJ may become a potential new treatment strategy.

Keywords: SARS-CoV-2; alkaline serine protease with an acidic pI (ASPNJ); antiviral agent; degradation of S protein; spike (S) protein.

Figure 1

Molecular weight comparison of S, S1, and RBD-mouseFc by SDS-PAGE and schematic diagram of S protein. (a) Lane 1; Bovine serum albumin (BSA), MW 66 kD. Lane 2; MW marker 1 (M1). Lane 3; RBD-mouseFc (Rm), MW~55 kD. Lane 4; S1 protein (S1), approximately 110 kD. Lane 5; Spike protein(S), approximately 150 kD. Lane 6; MW marker 2 (M2). (b) S protein contains signal peptide (1–12 aa), S1 protein (13–685 aa) and S2 protein (686–1273 aa). RBD (319–540 aa) was a domain in S1 protein. The C-terminal sequence of S1 was 682RRAR, which was a specific structure motif, termed as C-end rule (CendR).

Figure 2

Degradation of S protein, S1 protein, and RBD-mouseFc by ASPNJ of different concentrations: (a) degradation of S; (b) degradation of S1; and (c) degradation of RBD-mouseFc (Rm). The concentration of S, S1, and Rm were all 10 μg/lane. A1–A5 were ASPNJ/lanes of different concentrations (A1–A5 were 50 (or 30), 1, 0.2, 0.04, 0.008 μg/lane, respectively). Each of the three proteins was degraded by ASPNJ, with a total volume of 25 μL, at 40 °C for 60 min, and the degradation effect was observed by running SDS-PAGE.

Figure 3

Degradation of S, S1, and Rm by ASPNJ for different incubation times: (a) degradation of S; (b) degradation of S1; (c) degradation of RBD-mouseFc (Rm); and (d) degradation of S, S1 and Rm by ASPNJ for 1 min. The concentrations of S, S1, and Rm were all 10 μg/lane. A1 and A2 were ASPNJ of different concentrations (A1–A2 was 30, 1 μg/lane). B: BSA. The three proteins were degraded with ASPNJ with a total volume of 25 μL, and degraded at 40 °C for 1, 15, and 30 to 60 min, respectively. The degradation effect was observed by running SDS-PAGE. The lowercase letter a in (d) represents the denatured ASPNJ, which was heated at 80 °C for 5 min. Both of A2 and a2 were 1 μg/lane.

Figure 4

The action of ASPNJ on BSA and the action of trypsin on S1 protein. (a) BSA was degraded with ASPNJ with a total volume of 25 μL and degraded at 40 °C for 60 min. A: ASPNJ of different concentrations (A1–A5 were 50, 1, 0.2, 0.04 and 0.008 μg/lane, respectively). B: BSA (10 μg/lane). (b) Separate degradation reactions of different concentrations of trypsin to S1 protein, with a total volume of 25 μL, were carried out at 40 °C for 60 min. (c) Individual degradation reactions of S1 protein by trypsin occurred at different incubation times from 1, 15, 30 to 60 min. S1 protein was 10 μg/lane. T: Trypsin at different concentrations (50, 1, 0.2, 0.04, 0.008, 100 and 5 μg/lane for T1–T7, respectively). The degradation effects were observed by running SDS-PAGE.