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

Authors:

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.
Citation: Tanikawa, T.; Kiba, Y.; Yu, J.;
Hsu, K.; Chen, S.; Ishii, A.; Yokogawa,
T.; Suzuki, R.; Inoue, Y.; Kitamura, M.
Degradative Effect of Nattokinase on
Spike Protein of SARS-CoV-2.
Molecules 2022,27, 5405. https://
doi.org/10.3390/molecules27175405
Academic Editor: Maria JoséU.
Ferreira
Received: 14 July 2022
Accepted: 23 August 2022
Published: 24 August 2022
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molecules
Article
Degradative Effect of Nattokinase on Spike Protein
of SARS-CoV-2
Takashi Tanikawa 1, *,† , Yuka Kiba 2,† , James Yu 3, Kate Hsu 3, Shinder Chen 3, Ayako Ishii 4, Takami Yokogawa 2,
Ryuichiro Suzuki 5, Yutaka Inoue 1and Masashi Kitamura 2, *
1Laboratory of Nutri-Pharmacotherapeutics Management, School of Pharmacy,
Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan
2Laboratory of Pharmacognocy, School of Pharmacy, Faculty of Pharmacy and Pharmaceutical Sciences,
Josai University, Saitama 350-0295, Japan
3Contek Life Science Co., Ltd., Taipei City 100007, Taiwan
4CellMark Japan, Tokyo 102-0071, Japan
5Laboratory of Natural Products & Phytochemistry, Department of Pharmaceutical Sciences,
Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan
*Correspondence: tanikawa@josai.ac.jp (T.T.); kitamura@josai.ac.jp (M.K.)
These authors contributed equally to this work.
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: SARS-CoV-2; nattokinase; COVID-19
1. Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has been spreading worldwide. The COVID-19 pandemic
has affected over 437 million people and caused more than 6.3 million deaths (https:
//covid19.who.int/, accessed on 4 July 2022). The entry of SARS-CoV-2 into host cells
is mediated by the transmembrane spike protein (S protein), which forms homotrimers
that extend from the viral envelope. The S protein is processed and activated by cellular
proteases including transmembrane serine protein 2 (TMPRSS2), cathepsin, and furin. It
comprises two functional subunits, S1 and S2; the S1 subunit of SARS-CoV-2 initiates virus-
receptor binding by interacting with the human host cell receptor angiotensin-converting
enzyme 2 (ACE2), and the S2 subunit participates in viral fusion with the target cell,
allowing viral entry [
1
]. The receptor-binding domain (RBD) in the S1 subunit is responsible
for binding to ACE2. S protein cleavage occurs at the boundary between the S1 and
S2 subunits.
Currently, many countries are involved in the development of vaccines to protect
against SARS-CoV-2 infection, thus, the number of SARS-CoV-2 infections has decreased.
However, numerous variants of SARS-CoV-2, including strains with mutated vaccine tar-
get epitopes, have been reported [
2
,
3
]. Vaccination may not completely protect against
Molecules 2022,27, 5405. https://doi.org/10.3390/molecules27175405 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 5405 2 of 7
SARS-CoV-2 infection because the number of patients with COVID-19 is increasing after vac-
cination. Therefore, it is important to develop novel treatments for
SARS-CoV-2 infections.
Natto is a popular traditional Japanese food made from soybeans fermented by Bacillus
subtilis var. natto. Nattokinase is found in natto [
4
] and is one of the most important extracel-
lular enzymes produced by B. subtilis var. natto [
5
]. Nattokinase consists of 275 amino acids
and is approximately 28 kDa [
6
,
7
]. Nattokinase inactivates plasminogen activator inhibitor-
1 and increases fibrinolysis [
8
]. It also decreases the plasma levels of fibrinogen, factor VII,
cytokines, and factor VIII [
9
]. Nattokinase has the highest clot-dissolving potency among
naturally known anticoagulants [
10
]. A clinical trial demonstrated that oral consumption
of nattokinase was not associated with any adverse effects [
11
]. Thus, nattokinase is now
considered an efficient, secure, and economical enzyme that has drawn central attention in
thrombolytic drug studies [
12
,
13
]. In addition, nattokinase is used in the treatment of some
tumors [14,15].
A recent study revealed that natto extract inhibits bovine herpesvirus 1 (BHV-1) and
SARS-CoV-2 infection [
16
]. These results indicate that natto extract protease might be
effective against SARS-CoV-2 infection. In this study, we aimed to investigate whether the
inhibition of SARS-CoV-2 infection by natto extract is caused by nattokinase derived from
B. subtilis var. natto.
2. Results and Discussion
2.1. Degradative Effects of Nattokinase on Spike Protein of SARS-CoV-2 In Vitro
We first investigated whether nattokinase in natto extract could degrade SARS-CoV-2
S protein. The S protein of SARS-CoV-2 plays an important role in the ACE2 receptor of
the host cell during the early stages of infection [
17
]. After mixing the S protein expres-
sion cell lysate with a 4-fold dilution series of nattokinase (32
µ
g/mL, 8
µ
g/mL,
2µg/mL
,
500 ng/mL
, 125 ng/mL, 31.25 ng/mL, and 7.8125 ng/mL), Western blotting was performed.
The full length of S protein (S1 and S2 subunits) and S2 subunit appeared as bands when
S protein expression cell lysate was incubated with D-PBS at nattokinase concentrations
of 500 ng/mL, 125 ng/mL, 31.25 ng/mL, and 7.8125 ng/mL (Figure 1A). Next, we ex-
amined whether nattokinase degrades the S protein in a time-dependent manner. The
lysate was then incubated with 1
µ
g/mL nattokinase for 10–180 min. The S protein of
SARS-CoV-2
was degraded by nattokinase after 60–180 min of incubation, but not after 10
and 30 min of incubation (Figure 1B). Thus, nattokinase degraded S protein in a dose- and
time-dependent manner.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 7
target epitopes, have been reported [2,3]. Vaccination may not completely protect against
SARS-CoV-2 infection because the number of patients with COVID-19 is increasing after
vaccination. Therefore, it is important to develop novel treatments for SARS-CoV-2 infec-
tions.
Natto is a popular traditional Japanese food made from soybeans fermented by Ba-
cillus subtilis var. natto. Nattokinase is found in natto [4] and is one of the most important
extracellular enzymes produced by B. subtilis var. natto [5]. Nattokinase consists of 275
amino acids and is approximately 28 kDa [6,7]. Nattokinase inactivates plasminogen acti-
vator inhibitor-1 and increases fibrinolysis [8]. It also decreases the plasma levels of fibrin-
ogen, factor VII, cytokines, and factor VIII [9]. Nattokinase has the highest clot-dissolving
potency among naturally known anticoagulants [10]. A clinical trial demonstrated that
oral consumption of nattokinase was not associated with any adverse effects [11]. Thus,
nattokinase is now considered an efficient, secure, and economical enzyme that has drawn
central attention in thrombolytic drug studies [12,13]. In addition, nattokinase is used in
the treatment of some tumors [14,15].
A recent study revealed that natto extract inhibits bovine herpesvirus 1 (BHV-1) and
SARS-CoV-2 infection [16]. These results indicate that natto extract protease might be ef-
fective against SARS-CoV-2 infection. In this study, we aimed to investigate whether the
inhibition of SARS-CoV-2 infection by natto extract is caused by nattokinase derived from
B. subtilis var. natto.
2. Results and Discussion
2.1. Degradative Effects of Nattokinase on Spike Protein of SARS-CoV-2 In Vitro
We first investigated whether nattokinase in natto extract could degrade SARS-CoV-
2 S protein. The S protein of SARS-CoV-2 plays an important role in the ACE2 receptor of
the host cell during the early stages of infection [17]. After mixing the S protein expression
cell lysate with a 4-fold dilution series of 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), Western blotting was performed. The
full length of S protein (S1 and S2 subunits) and S2 subunit appeared as bands when S
protein expression cell lysate was incubated with D-PBS at nattokinase concentrations of
500 ng/mL, 125 ng/mL, 31.25 ng/mL, and 7.8125 ng/mL (Figure 1A). Next, we examined
whether nattokinase degrades the S protein in a time-dependent manner. The lysate was
then incubated with 1 µg/mL nattokinase for 10180 min. The S protein of SARS-CoV-2
was degraded by nattokinase after 60180 min of incubation, but not after 10 and 30 min
of incubation (Figure 1B). Thus, nattokinase degraded S protein in a dose- and time-de-
pendent manner.
Figure 1. Cont.
Molecules 2022,27, 5405 3 of 7
Molecules 2022, 27, x FOR PEER REVIEW 3 of 7
Figure 1. (A) Degradative effects of nattokinase in dose-dependent manner. Serial diluted nattoki-
nase (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 sub-
units) 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 pro-
tein) + 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.
To confirm whether the degradative effect of nattokinase is due to enzymatic activity,
nattokinase was treated with heating or a protease inhibitor cocktail. When nattokinase
was heated at 100 °C for 5 min, the degradative effect of nattokinase was lost (Figure 1C,
lane 6). Furthermore, the loss of the S protein bands by nattokinase was blocked when
protease inhibitors were added (Figure 1C, lanes 4 and 5). Compared with protein inhib-
itor cocktail I, protein cocktail III, which consisted of AEBSF HCl (4-(2-Aminoethyl) ben-
zenesulfonyl fluoride hydrochloride), aprotinin, which is an irreversible serine protease
inhibitor, and leupeptin, which is a cysteine-protease, clearly blocked nattokinase activity.
Nattokinase has the same conserved amino acids, Ser-His-Asp (Asp32, His64, and Ser221),
which are members of the subtilisin family of serine proteases [6,18]. The crystal structure
of nattokinase is nearly identical to that of subtilisin E from B. subtilis DB104 [19]. This
result is consistent with that of a previous report that nattokinase is a serine protease. We
also assessed the degradative effects of nattokinase using cell lysates expressing the RBD
and ACE2. When 7.5 µg/mL of nattokinase and cell lysate were incubated, the bands of
RBD and ACE2 were lost (Figure 1D).
Figure 1.
(
A
) Degradative effects of nattokinase in dose-dependent manner. Serial diluted nattokinase
(32
µ
g/mL, 8
µ
g/mL, 2
µ
g/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
1µg/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
(
5µg/mL
); lane 4: HEK293 (S protein) + nattokinase (
5µg/mL
) + Protease inhibitor I; lane 5: HEK293
(S protein) + nattokinase (
5µg/mL
) + Protease inhibitor III; lane 6: HEK293 (S protein) + heat-treated
nattokinase (5
µ
g/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
µ
g/mL) and heat-treated nattokinase (7.5
µ
g/mL) and Western
blotting was performed.
To confirm whether the degradative effect of nattokinase is due to enzymatic activity,
nattokinase was treated with heating or a protease inhibitor cocktail. When nattokinase was
heated at 100
C for 5 min, the degradative effect of nattokinase was lost (
Figure 1C, lane 6
).
Furthermore, the loss of the S protein bands by nattokinase was blocked when protease
inhibitors were added (Figure 1C, lanes 4 and 5). Compared with protein inhibitor cocktail
I, protein cocktail III, which consisted of AEBSF HCl (4-(2-Aminoethyl) benzenesulfonyl
fluoride hydrochloride), aprotinin, which is an irreversible serine protease inhibitor, and
leupeptin, which is a cysteine-protease, clearly blocked nattokinase activity. Nattokinase
has the same conserved amino acids, Ser-His-Asp (Asp
32
, His
64
, and Ser
221
), which are
members of the subtilisin family of serine proteases [
6
,
18
]. The crystal structure of nat-
tokinase is nearly identical to that of subtilisin E from B. subtilis DB104 [
19
]. This result
is consistent with that of a previous report that nattokinase is a serine protease. We also
assessed the degradative effects of nattokinase using cell lysates expressing the RBD and
ACE2. When 7.5
µ
g/mL of nattokinase and cell lysate were incubated, the bands of RBD
and ACE2 were lost (Figure 1D).
Molecules 2022,27, 5405 4 of 7
2.2. Degradative Effects of Nattokinase on Spike Protein of SARS-CoV-2 on the Transfected
Cell Surface
Next, we examined whether nattokinase degrades the S protein on the transfected
cell surface. The S protein was transfected with the HEK293 cells. The transfected cells
were incubated with nattokinase for 9 h. The S protein on the cell surface was detected
using an anti-S protein antibody without cell permeabilization (Figure 2A). The S protein
was detected in the transfected cells. When transfected cells were treated with nattokinase,
the S protein on the cell surface decreased. When cells were treated with 25
µ
g/mL
and
2.5 µg/mL
nattokinase, the ratio of S protein-positive area to nucleus-positive area
decreased by approximately 0.3 and 0.7, respectively (Figure 2B). The degradative effect of
nattokinase was observed when there was no cytotoxicity (Figure 2C). Western blotting
analysis showed that the quantity of total S protein did not change among nattokinase and
control treatments (Supplemental Figure; Figure S1). These results indicate that nattokinase
would degrade the S protein of SARS-CoV-2 in the non-toxic concentration range.
Molecules 2022, 27, x FOR PEER REVIEW 4 of 7
2.2. Degradative Effects of Nattokinase on Spike Protein of SARS-CoV-2 on the Transfected Cell
Surface
Next, we examined whether nattokinase degrades the S protein on the transfected
cell surface. The S protein was transfected with the HEK293 cells. The transfected cells
were incubated with nattokinase for 9 h. The S protein on the cell surface was detected
using an anti-S protein antibody without cell permeabilization (Figure 2A). The S protein
was detected in the transfected cells. When transfected cells were treated with nattokinase,
the S protein on the cell surface decreased. When cells were treated with 25 µg/mL and
2.5 µg/mL nattokinase, the ratio of S protein-positive area to nucleus-positive area de-
creased by approximately 0.3 and 0.7, respectively (Figure 2B). The degradative effect of
nattokinase was observed when there was no cytotoxicity (Figure 2C). Western blotting
analysis showed that the quantity of total S protein did not change among nattokinase
and control treatments (Supplemental Figure; Figure S1). These results indicate that nat-
tokinase would degrade the S protein of SARS-CoV-2 in the non-toxic concentration
range.
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 immu-
nofluorescent analysis was performed. S protein on the cell surface was stained with anti-spike pro-
tein 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 var-
iance (ANOVA) with Tukeys 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.
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
µ
g/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.
Molecules 2022,27, 5405 5 of 7
In this study, we showed that the protease activity of nattokinase contributes to the
degradation of S protein. Nattokinase has a degrading effect on not only S proteins but also
ACE2 in host cells. The protease specificity of nattokinase would be low, because GAPDH,
a housekeeping protein, was also degraded simultaneously in the in-vitro evaluation of
nattokinase mixed with cell lysate (Supplemental Figure; Figure S2). On the other hand,
when added to cells, it does not show any effect on cell viability and is expected to act
as a protective agent on the cell surface. Further analysis of the degradation products of
nattokinase using mass spectrometry is needed for understanding the proteolysis effects.
Nattokinase possesses the potent degradation activity for SARS-CoV-2 S protein
and has also been shown to exert anti-atherosclerotic, lipid-lowering, antihypertensive,
antithrombotic, fibrinolytic, neuroprotective, antiplatelet, and anticoagulant effects [
20
].
Patients with hypertension and cardiovascular comorbidities can easily get very sick from
COVID-19 [
21
]. Due to the emergence of numerous variants of SARS-CoV-2 including
strains with mutated vaccine target epitopes, vaccination alone may not completely protect
against SARS-CoV-2 infection. Nattokinase and natto extracts have the potential to be
developed as a new generation of drug for the prevention and treatment of COVID-19.
3. Materials and Methods
3.1. Materials
Nattokinase was obtained from Contek Life Science Co., Ltd. (Taipei City, Taiwan).
The nattokinase activity was 60,000 FU/g (FU, fibrinolysis unit). Protease inhibitor cocktail
sets I and III were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka,
Japan). The expression plasmid (pcDNA3.1-SARS2-Spike C9 with tag at the C-terminal,
pcDNA3.1-hACE2, and pcDNA3-SARS-CoV-2-S-RBD-sfGFP) was purchased from Ad-
dgene (Watertown, MA, USA). HEK293 (JCRB9068) cells were obtained from the JCRB Cell
Bank (Osaka, Japan).
3.2. Cell Culture and Western Blotting
HEK293 cells were cultured at a density of 3.5
×
10
5
cells/mL in DMEM supplemented
with 10% FBS, L-glutamine, 100 U/mL penicillin, and 100
µ
g/mL streptomycin overnight.
The cells were transfected with each plasmid (pcDNA3.1-SARS2-Spike, pcDNA3-SARS-
CoV-2-S-RBD-sfGFP, or pcDNA3.1-hACE2) and incubated for 22 h. After incubation, the
cultured cells were scraped and washed with ice-cold Dulbecco’s phosphate-buffered saline
(D-PBS). Cell counting was performed and xTractor buffer (Takara Bio Inc., Shiga, Japan)
was added to the cell precipitate. The cell lysates were centrifuged at 1300
×
gfor 10 min
at 4
C and the supernatant was transferred to new tubes and stored at
80
C until
use. The protein concentration was determined by bicinchoninic acid (BCA) protein assay
using a BCA assay kit (Takara). Ten microliters of nattokinase and 10
µ
L of cell lysate
(
1µ µg/mL
) were incubated at 37
C for 1 h. When the effects of protease inhibitors were
used, protease inhibitor cocktail sets I and III were diluted 10-fold with D-PBS, and a 10
µ
L
protease inhibitor cocktail solution was added to the mixture of nattokinase and cell lysate.
Equal volumes of the reaction mixture were loaded and Western blotting was performed.
The primary antibodies included anti-rhodopsin (C9) mouse monoclonal antibody (1D4)
(Santa Cruz Biotechnology, Dallas, TX, USA), anti-GAPDH mouse monoclonal antibody
(FUJIFILM Wako), anti-GFP tag mouse monoclonal antibody (Proteintech, Rosemont,
IL, USA), and anti-ACE2 antibody (Proteintech). Secondary antibodies include HRP-
conjugated goat anti-mouse antibody (Proteintech).
3.3. Immunofluorescence Assay
HEK293 cells were cultured at a density of 3.5
×
10
5
cells/mL in an 8-well chamber in
DMEM supplemented with 10% FBS, L-glutamine, 100 U/mL penicillin, and 100
µ
g/mL
streptomycin. The cells were transfected with pcDNA3.1-SARS2-Spike and incubated for
9 h. After incubation, the cells were treated with the samples, incubated for 13 h, and
fixed in 4% paraformaldehyde for 30 min. After incubation with SARS-CoV/
SARS-CoV-2
Molecules 2022,27, 5405 6 of 7
spike monoclonal antibody (1A9) (GeneTex, CA, USA) for 1 h, it was incubated with
Cy3–conjugated goat anti-mouse antibody for 1 h. The slides were stained with DAPI
Fluoromount-G and observed using a fluorescence microscope (BZ-X710, Keyence, Os-
aka, Japan). S protein-positive and nucleus-positive areas were calculated using BZ-X710
attached analysis software (BZ-X Analyzer). Cell viability was assessed using the 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were cul-
tivated in 24-well culture plates. After incubation at 37
C for 24 h, samples were added
to each well and incubated for another 13 h. Cells were suspended in 500
µ
L of DMEM
containing 500
µ
g/mL MTT. After incubation for 3 h at 37
C, 500
µ
L isopropanol contain-
ing
4 mM
HCl was added to dissolve MTT formazan. The absorbance was measured at
570 nm using a microplate reader.
4. Conclusions
In this study, we demonstrated that nattokinase, a serine protease, degrades the S
protein of SARS-CoV-2. To investigate whether nattokinase contained in natto extract could
inhibit SARS-CoV-2 infection, we analyzed S protein degradation by mixing the S protein
expression cell lysate and nattokinase in a dose- and time-dependent manner. The RBD of
the S protein binds to the membrane-distal portion of the ACE2 protein. Natto extract has
been reported to inhibit SARS-CoV-2 infection in Vero E6 cells via RBD degradation [
16
].
We demonstrated that S protein degradation by nattokinase was blocked by heat or protein-
inhibitor treatments. Our data suggest that the protease activity of nattokinase plays a
crucial role in S protein degradation. Taken together, these findings support the notion that
the inhibition of SARS-CoV-2 infection by natto extract was due to S protein degradation
by nattokinase. Thus, our data indicated that nattokinase and natto extracts have potential
effects on the inhibition of SARS-CoV-2 host cell entry via S protein degradation.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules27175405/s1, Figure S1: Effect of nattokinase addition
to S protein expressing cell culture medium was evaluated by Western blotting; Figure S2: Degradative
effects of GAPDH in vitro.
Author Contributions:
Conceptualization and supervision, T.T., J.Y., K.H., S.C., A.I., T.Y., R.S., Y.I.
and M.K.; Methodology, all authors; Western blot and immunofluorescent analyses, T.T., Y.K. and
M.K.; writing—original draft preparation, T.T. and M.K.; writing—review and editing, all authors.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data used to support these findings have been included in this
article. Additional information is available from the corresponding authors upon request.
Conflicts of Interest: The authors declare no conflict of interest.
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... Thirdly, OND intervention twelve months post-acute infection might have contributed, considering the decreasing receptor binding of S protein over the disease course [12]. Additionally, the prolonged use of nattokinase, a time-dependent protease inhibitor, and nicotine patches four months prior could influence S protein inhibition and receptor binding, respectively [42,43]. ...
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Long COVID, potentially emerging post COVID-19 infection, involves extreme health challenges. Based on current literature in the field, we propose a novel approach to Long COVID treatment based on epipharyngeal abrasive therapy targeting ostia of the oral and nasal mucosa, having been identified for the first time. The presented case report documents the application of innovative oronasal drainage (OND), a novel treatment integrating physiological, biochemical, and fluid mechanical components simultaneously. OND led to remarkable improvements and even remissions of various symptoms, along with enhanced hand blood circulation. While the case suggests potential efficacy in Long COVID therapy, acknowledging inherent limitations is essential and its impact needs further validation through clinical trials.
... . While the proteolytic specificity of nattokinase, as an alkaline serine protease [44,73,273], is surprisingly underexplored, beyond a broad similarity to that of plasmin [44,274] (and nattokinase can even degrade spike protein [275] and certain 'classical' amyloids [276][277][278]), the question arises as to whether or not nattokinase can degrade the amyloid 'fibrinaloid' form of microclots. The purposes of this paper are (i) to describe an efficient, quantitative, automated microscopic method that can be used to determine the size and number of amyloid microclots and any time-dependent changes therein, and thus (ii) to assess any such nattokinase-induced degradation of the microclots, concluding that nattokinase can indeed degrade fibrinaloid microclots effectively. ...
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Nattokinase, from the Japanese fermented food natto, is a protease with fibrinolytic activity that can thus degrade conventional blood clots. In some cases, however, including in Long COVID, fibrinogen can polymerise into an anomalous amyloid form to create clots that are resistant to normal fibrinolysis and that we refer to as fibrinaloid microclots. These can be detected with the fluorogenic stain thioflavin T. We describe an automated microscopic technique for the quantification of fibrinaloid microclot formation, which also allows the kinetics of their formation and aggregation to be recorded. We also here show that recombinant nattokinase is effective at degrading the fibrinaloid microclots in vitro. Flow conditions, mimicked by shaking, increase the size of the clots via aggregation. Overall, this work adds to the otherwise largely anecdotal evidence, that we review, that nattokinase might be anticipated to have value as part of therapeutic treatments for individuals with Long COVID and related disorders that involve fibrinaloid microclots.
... 149 Finally, the fibrinolytic enzyme nattokinase can be used in conjunction with lutein, as it has recently been shown to effectively degrade the spike protein of SARS-CoV-2. 150 ...
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Lutein, a plant-derived xanthophyl-carotenoid, is an exceptional antioxidant and anti-inflammatory constituent found in food. High dietary intake of lutein is beneficial against eye disease, improves cardiometabolic health, protects from neurodegenerative diseases, and is beneficial for liver, kidney, and respiratory health. Lutein protects against oxidative and nitrosative stress, both of which play a major role in long COVID and mRNA vaccination injury syndromes. Lutein is an important natural agent for therapeutic use against oxidative and nitrosative stress in chronic illnesses such as cardiovascular and neurodegenerative diseases and cancer. It can also potentially inhibit spike protein-induced inflammation. Rich dietary supplementation of lutein, naturally derived in non-biodegradable Extra Virgin Olive Oil (EVOO), can most optimally be used against oxidative and nitrosative stress during post-COVID and mRNA vaccination injury syndromes. Due to its high oleic acid (OA) content, EVOO supports optimal absorption of dietary lutein. The main molecular pathways by which the SARS-CoV-2 spike protein induces pathology, nuclear factor kappa-light-chain-enhancer activated B cells (NF-κB) and activated protein (AP)-1, can be suppressed by lutein. Synergy with other natural compounds for spike protein detoxification is likely. Open Peer Review Approval Status 1 2 version 3 (revision)
... 149 Finally, the fibrinolytic enzyme nattokinase can be used in conjunction with lutein, as it has recently been shown to effectively degrade the spike protein of SARS-CoV-2. 150 ...
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Lutein, a plant-derived xanthophyl-carotenoid, is an exceptional antioxidant and anti-inflammatory constituent found in food. High dietary intake of lutein is beneficial against eye disease, improves cardiometabolic health, protects from neurodegenerative diseases, and is beneficial for liver, kidney, and respiratory health. Lutein protects against oxidative and nitrosative stress, both of which play a major role in long COVID and mRNA vaccination injury syndromes. Lutein is an important natural agent for therapeutic use against oxidative and nitrosative stress in chronic illnesses such as cardiovascular and neurodegenerative diseases and cancer. It can also potentially inhibit spike protein-induced inflammation. Rich dietary supplementation of lutein, naturally derived in non-biodegradable Extra Virgin Olive Oil (EVOO), can most optimally be used against oxidative and nitrosative stress during post-COVID and mRNA vaccination injury syndromes. Due to its high oleic acid (OA) content, EVOO supports optimal absorption of dietary lutein. The main molecular pathways by which the SARS-CoV-2 spike protein induces pathology, nuclear factor kappa-light-chain-enhancer activated B cells (NF-κB) and activated protein (AP)-1, can be suppressed by lutein. Synergy with other natural compounds for spike protein detoxification is likely.
... The production of natto takes place via fermentation following the addition of the bacterium Bacillus natto to soybeans, resulting in the production of the nattokinase enzyme [63]. Nattokinase has been demonstrated to exert several health effects, including anti-atherosclerotic, lipidlowering, antihypertensive, antithrombotic, fibrinolytic, neuroprotective, antiplatelet, and anticoagulant effects [68]. ...
... In a study by Oba and colleagues, natto extract was shown to inhibit bovine herpesvirus 1 (BHV-1) and SARS-CoV-2 infection [59]. Moreover, the inhibition of SARS-CoV-2 infection by natto extract was achieved by the degradation of S protein by NK [60]. Given the observed health benefits of NK, this natural, safe, effective, and inexpensive dietary supplement is a promising agent for the treatment or prevention of CVDs and beyond [55]. ...
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The use of non-vitamin K antagonist oral anticoagulants (NOACs) has brought a significant progress in the management of cardiovascular diseases, considered clinically superior to vitamin K antagonists (VKAs) particularly in the prevention and treatment of thromboembolic events. In addition, numerous advantages such as fixed dosing, lack of laboratory monitoring, and fewer food and drug-to-drug interactions make the use of NOACs superior to VKAs. While NOACs are synthetic drugs prescribed for specific conditions, nattokinase (NK) is a natural enzyme derived from food that has potential health benefits. Various experimental and clinical studies reported the positive effects of NK on the circulatory system, including the thinning of blood and the dissolution of blood clots. This enzyme showed not only fibrinolytic activity due to its ability to degrade fibrin, but also an affinity as a substrate for plasmin. Recent studies have shown that NK has additional cardioprotective effects, such as antihypertensive and anti-atherosclerotic effects. In this narrative review, we presented the cardioprotective properties of two different approaches that go beyond anticoagulation: NOACs and NK. By combining evidence from basic research with clinical findings, we aim to elucidate the comparative cardioprotective efficacy of these interventions and highlight their respective roles in modern cardiovascular care.
... 144 Finally, the fibrinolytic enzyme nattokinase can be used in conjunction with lutein, as it has recently been shown to effectively degrade the spike protein of SARS-CoV-2. 145 ...
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... 139 Finally, the fibrinolytic enzyme nattokinase can be used in conjunction with lutein, as it has recently been shown to effectively degrade the spike protein of SARS-CoV-2. 140 ...
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Lutein, a plant-derived xanthophyl-carotenoid, is an exceptional antioxidant and anti-inflammatory constituent found in food. High dietary intake of lutein is beneficial against eye disease, improves cardiometabolic health, protects from neurodegenerative diseases, and is beneficial for liver, kidney, and respiratory health. Lutein protects against oxidative and nitrosative stress, both of which play a major role in post-COVID and mRNA vaccination injury syndromes. Lutein is an important natural agent for therapeutic use against oxidative and nitrosative stress in chronic illnesses such as cardiovascular and neurodegenerative diseases and cancer. It can also potentially inhibit spike protein-induced inflammation. Rich dietary supplementation of lutein, naturally derived in non-biodegradable Extra Virgin Olive Oil (EVOO), can most optimally be used against oxidative and nitrosative stress during post-COVID and mRNA vaccination injury syndromes. Due to its high oleic acid (OA) content, EVOO supports optimal absorption of dietary lutein. The main molecular pathways by which the SARS-CoV-2 spike protein induces pathology, nuclear factor kappa-light-chain-enhancer activated B cells (NF-κB) and activated protein (AP)-1, can be suppressed by lutein. Synergy with other natural compounds for spike protein detoxification is likely.
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- this is an update v2 of preprint v1 (v1: feb2024, doi: 10.13140/RG.2.2.16142.33600) - illustrative and aesthetic enhancements in some figures. - restructuring and improvement of some passages of the content
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Cardiovascular disease (CVD) is the leading cause of death in the world and our approach to the control and management of CVD mortality is limited. Nattokinase (NK), the most active ingredient of natto, possesses a variety of favourable cardiovascular effects and the consumption of Natto has been linked to a reduction in CVD mortality. Recent research has demonstrated that NK has potent fibrinolytic activity, antihypertensive, anti-atherosclerotic, and lipid-lowering, antiplatelet, and neuroprotective effects. This review covers the major pharmacologic effects of NK with a focus on its clinical relevance to CVD. It outlines the advantages of NK and the outstanding issues pertaining to NK pharmacokinetics. Available evidence suggests that NK is a unique natural compound that possesses several key cardiovascular beneficial effects for patients with CVD and is therefore an ideal drug candidate for the prevention and treatment of CVD. Nattokinase is a promising alternative in the management of CVD.
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Nattokinase (NK, EC 3.4.21.62) is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of Hepatocellular carcinoma (HCC), a principal malignant tumor that causes morbidity and mortality worldwide. Crudes extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and Vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and Vimentin in the NCE groups. Taken together, these data demonstrate that NCE from the Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.
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Thrombotic disease has become one of the leading causes of mortality among humans globally. Nattokinase (NK), a novel thrombolytic agent, has attracted the attention of researchers. However, NK is a serine protease that is vulnerable to environmental effects resulting in its inactivation. In this study, polylysine dendrimer (PLLD) was synthesized via divergence-convergence method, and a series of NK/PLLD nanocomposites with different molar ratio was prepared. In addition, NK was successfully incorporated into the cores of PLLD G4 through hydrogen bonds and van der Waals forces. In NK/PLLD nanocomposites, when the molar ratio of NK to PLLD is 1:30, a high relative enzyme activity level (up to 117%) was achieved and was more stable at different temperatures and pH than free NK. In in vitro thrombolysis experiment, compared with free NK, NK/PLLD nanocomposites could control the release of NK. The thrombolysis rate of NK/PLLD nanocomposites reached 50% at 12 h, which can effectively avoid other complications such as hemorrhage. Interestingly, NK/PLLD nanocomposites with positive charge can penetrate into the negatively charged thrombus via electrostatic interaction, thus providing a good thrombolytic effect. Hemolysis and MTT experiments show that PLLD nanomaterials can serve as ideal carriers of protein drugs. This article is protected by copyright. All rights reserved.