Effect of β-chitosan on the binding interaction between SARS-CoV-2 S-RBD and ACE2

Abstract

SARS-CoV-2 invades human respiratory epithelial cells via an interaction between its spike RBD protein (SARS-CoV-2 S-RBD) and the host cell receptor angiotensin converting enzyme II (ACE2). Blocking this interaction provides a potent approach to preventing and controlling SARS-CoV-2 infection. In this work, the ability of β-chitosan to block the binding interaction between SARS-CoV-2 S-RBD and ACE2 was investigated. The inhibitory effect of β-chitosan on inflammation induced by the SARS-CoV-2 S-RBD was also studied. Native-PAGE analysis indicated that β-chitosan could bind with ACE2 and the SARS-CoV-2 S-RBD and a conjugate of β-chitosan and ACE2 could no longer bind with the SARS-CoV-2 S-RBD. HPLC analysis suggested that a conjugate of β-chitosan and the SARS-CoV-2 S-RBD displayed high binding affinity without dissociation under high pressure (40 MPa) compared with that of β-chitosan and ACE2. Furthermore, immunofluorescent staining of Vero E6 cells and lungs from hACE2 mice showed that the presence of β-chitosan prevented SARS-CoV-2 S-RBD from binding to ACE2. Meanwhile, β-chitosan could dramatically suppress the inflammation caused by the presence of the SARS-CoV-2 S-RBD both in vitro and vivo . Moreover, the decreased expression of ACE2 caused by β-chitosan treatment was restored by addition of TAPI-1, an inhibitor of the transmembrane protease ADAM17. Our findings demonstrated that β-chitosan displays an antibody-like function capable of neutralizing the SARS-CoV-2 S-RBD and effectively preventing the binding of the SARS-CoV-2 S-RBD to ACE2. Moreover, ADAM17 activation induced by β-chitosan treatment can enhance the cleavage of the extracellular domain of ACE2, releasing the active ectodomain into the extracellular environment, which can prevent the binding, internalization, and degradation of ACE2 bound to the SARS-CoV-2 S-RBD and thus diminish inflammation. Our study provides an alternative avenue for preventing SARS-CoV-2 infection using β-chitosan.

SciScore for 10.1101/2020.07.31.229781: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: Ethics statement: All procedures in this study involving animals were reviewed and approved by the Minnan Normal University Animal Ethics and Welfare Committee (MNNU-AEWC-2020001).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Mouse experiments: Specific pathogen-free, 8-week-old male transgenic hACE2 mice and WT male mice (C57BL/6 background) were purchased from the experimental animal center of GemPharmatech (Nanjing, China).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Antibodies and reagents: Anti-His-Tag, anti-p-IKK, anti-p-JNK, and anti-p-c-Jun antibodies, …

SciScore for 10.1101/2020.07.31.229781: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: Ethics statement: All procedures in this study involving animals were reviewed and approved by the Minnan Normal University Animal Ethics and Welfare Committee (MNNU-AEWC-2020001).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Mouse experiments: Specific pathogen-free, 8-week-old male transgenic hACE2 mice and WT male mice (C57BL/6 background) were purchased from the experimental animal center of GemPharmatech (Nanjing, China).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Antibodies and reagents: Anti-His-Tag, anti-p-IKK, anti-p-JNK, and anti-p-c-Jun antibodies, FITC-conjugated donkey anti-mouse IgG, and Cy3-conjugated donkey anti-rabbit IgG were purchased from Affinity Biosciences, Inc. (Cincinnati, OH, USA).	Anti-His-Tag, suggested: None Anti-His-Tag suggested: None anti-p-IKK, anti-p-JNK, suggested: None anti-p-c-Jun suggested: (Santa Cruz Biotechnology Cat# sc-822, RRID:AB_627262) Cy3-conjugated donkey anti-rabbit IgG suggested: None
Anti-ACE2 antibody, HRP-conjugated anti-mouse IgG, and HRP-conjugated anti-rabbit IgG were obtained from Abcam (Cambridge, UK).	anti-mouse IgG suggested: None anti-rabbit IgG suggested: None
The following primary antibodies His-Tag (1:200, Affinity, T0009, USA) and ACE2 (1:200, Abcam, ab15348, UK) were used.	ACE2 suggested: (Abcam Cat# ab15348, RRID:AB_301861)
The membrane was then blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies (including anti-ACE2, anti-p-IKK, anti-p-JNK, anti-p-c-Jun, and anti-β-actin antibodies) overnight at 4°C.	anti-p-IKK, anti-p-JNK, anti-p-c-Jun, suggested: None anti-β-actin suggested: None
The membrane was then washed with Tris buffered saline buffer with Tween 20 (TBST) for three times and incubated with secondary antibody (HRP-conjugated anti-IgG) for 1 h.	anti-IgG suggested: None
Experimental Models: Cell Lines
Sentences	Resources
8 μM TAPI-1 was added to Vero E6 cells 30 min in advance of any additional treatment conditions.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Mouse experiments: Specific pathogen-free, 8-week-old male transgenic hACE2 mice and WT male mice (C57BL/6 background) were purchased from the experimental animal center of GemPharmatech (Nanjing, China).	C57BL/6 suggested: None
Software and Algorithms
Sentences	Resources
Antibodies and reagents: Anti-His-Tag, anti-p-IKK, anti-p-JNK, and anti-p-c-Jun antibodies, FITC-conjugated donkey anti-mouse IgG, and Cy3-conjugated donkey anti-rabbit IgG were purchased from Affinity Biosciences, Inc. (Cincinnati, OH, USA).	Affinity Biosciences suggested: None
Mouse experiments: Specific pathogen-free, 8-week-old male transgenic hACE2 mice and WT male mice (C57BL/6 background) were purchased from the experimental animal center of GemPharmatech (Nanjing, China).	GemPharmatech suggested: (GemPharmatech, RRID:SCR_017239)
The protein signals were evaluated by integrated option density (IOD) using Image J software (National Institutes of Health, Bethesda, MD, USA).	Image J suggested: (ImageJ, RRID:SCR_003070)
Statistical analysis: All data were analyzed with GraphPad Prism 8.0 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Effect of β-chitosan on the binding interaction between SARS-CoV-2 S-RBD and ACE2

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