Degradative Effect of Nattokinase on Spike Protein of SARS-CoV-2

Abstract

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.

Keywords: COVID-19; SARS-CoV-2; nattokinase.

Figure 1

(A) Degradative effects of nattokinase in dose-dependent manner. Serial diluted nattokinase (32 µg/mL, 8 µg/mL, 2 µg/mL, 500 ng/mL, 125 ng/mL, 31.25 ng/mL, and 7.8125 ng/mL) were mixed with S protein expression cell lysate and incubated. Full length of S protein (S1 and S2 subunits) and S2 subunit were detected as upper and lower bands, respectively. Ratio of total S was indicated as the relative quantity of S protein (S protein + S2 protein). (B) Degradative effects of nattokinase in time-dependent manner. S protein expression cell lysate was incubated with 1 µg/mL nattokinase for 0, 10, 30, 60, 120, and 180 min. (C) Effects of heating treatment or protease inhibitors. Lane 1: HEK293 lysate; lane 2: HEK293 lysate (S protein); lane 3: HEK293 (S protein) + nattokinase (5 µg/mL); lane 4: HEK293 (S protein) + nattokinase (5 µg/mL) + Protease inhibitor I; lane 5: HEK293 (S protein) + nattokinase (5 µg/mL) + Protease inhibitor III; lane 6: HEK293 (S protein) + heat-treated nattokinase (5 µg/mL). (D) Degradative effect on RBD of S protein and ACE2. RBD of S protein and ACE2 coding plasmids were transfected with HEK293 cells, respectively. Cell lysates were incubated with nattokinase (7.5 µg/mL) and heat-treated nattokinase (7.5 µg/mL) and Western blotting was performed.

Figure 1

(A) Degradative effects of nattokinase in dose-dependent manner. Serial diluted nattokinase (32 µg/mL, 8 µg/mL, 2 µg/mL, 500 ng/mL, 125 ng/mL, 31.25 ng/mL, and 7.8125 ng/mL) were mixed with S protein expression cell lysate and incubated. Full length of S protein (S1 and S2 subunits) and S2 subunit were detected as upper and lower bands, respectively. Ratio of total S was indicated as the relative quantity of S protein (S protein + S2 protein). (B) Degradative effects of nattokinase in time-dependent manner. S protein expression cell lysate was incubated with 1 µg/mL nattokinase for 0, 10, 30, 60, 120, and 180 min. (C) Effects of heating treatment or protease inhibitors. Lane 1: HEK293 lysate; lane 2: HEK293 lysate (S protein); lane 3: HEK293 (S protein) + nattokinase (5 µg/mL); lane 4: HEK293 (S protein) + nattokinase (5 µg/mL) + Protease inhibitor I; lane 5: HEK293 (S protein) + nattokinase (5 µg/mL) + Protease inhibitor III; lane 6: HEK293 (S protein) + heat-treated nattokinase (5 µg/mL). (D) Degradative effect on RBD of S protein and ACE2. RBD of S protein and ACE2 coding plasmids were transfected with HEK293 cells, respectively. Cell lysates were incubated with nattokinase (7.5 µg/mL) and heat-treated nattokinase (7.5 µg/mL) and Western blotting was performed.

Figure 2

(A) Degradative effect of nattokinase on S protein on the cell surface. Spike-pcDNA3.1 was transfected with HEK293 cells and incubated for 9 h. After incubation, nattokinase (25 and 2.5 µg/mL) were added to culture medium and further incubated for 13 h. Cells were fixed and immunofluorescent analysis was performed. S protein on the cell surface was stained with anti-spike protein antibody (Red) and nucleus was stained with DAPI (Blue). (B) Ratio of S protein area to nucleus positive area. Three images per sample were captured and S protein/nucleus positive areas were calculated. Data are shown as mean + SD, and p-value was determined by one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using R software (R-3.3.3 for windows) (** p < 0.01; *** p < 0.001). (C) Cell viability was evaluated by MTT assay. Indicated nattokinase was added to culture medium and incubated for 13 h; MTT assay was performed.