key: cord-0877008-bzq1xxqb
authors: Wang, Cheng; Wang, Shaobo; Li, Daixi; Zhao, Xia; Han, Songling; Wang, Tao; Zhao, Gaomei; Chen, Yin; Chen, Fang; Zhao, Jianqi; Wang, Liting; Sun, Wei; Huang, Yi; Su, Yongping; Wei, Dongqing; Zhao, Jinghong; Wang, Junping
title: Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2
date: 2020-03-31
journal: bioRxiv
DOI: 10.1101/2020.03.29.013490
sha: 0c8ac295acf684a385eb45d0b37710affa3a9e85
doc_id: 877008
cord_uid: bzq1xxqb

The burgeoning epidemic caused by novel coronavirus 2019 (2019-nCoV) is currently a global concern. Angiotensin-converting enzyme-2 (ACE2) is a receptor of 2019-nCoV spike 1 protein (S1) and mediates viral entry into host cells. Despite the abundance of ACE2 in small intestine, few digestive symptoms are observed in patients infected by 2019-nCoV. Herein, we investigated the interactions between ACE2 and human defensins (HDs) specifically secreted by intestinal Paneth cells. The lectin-like HD5, rather than HD6, bound ACE2 with a high affinity of 39.3 nM and weakened the subsequent recruitment of 2019-nCoV S1. The cloak of HD5 on the ligand-binding domain of ACE2 was confirmed by molecular dynamic simulation. A remarkable dose-dependent preventive effect of HD5 on 2019-nCoV S1 binding to intestinal epithelial cells was further evidenced by in vitro experiments. Our findings unmasked the innate defense function of lectin-like intestinal defensin against 2019-nCoV, which may provide new insights into the prevention and treatment of 2019-nCoV infection.

Coronavirus owns enveloped positive-stranded RNA encoding the spike (S) protein, envelope protein, membrane protein, and nucleoprotein. S protein is composed of 1160-1400 amino acids and presents as a trimer before membrane fusion 6 . Many S proteins can be divided into S1 and S2 subunits after degradation by protease 7 . The S1 subunit contains a receptor-binding domain (RBD) and devotes to the initial viral attachment via recognizing a variety of host cell receptors, including aminopeptidase N (APN), angiotensin-converting enzyme-2 (ACE2), dipeptidyl peptidase 4 (DPP4), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and sugar 8, 9, 10 . Since the sequence of 2019-nCoV S protein is 76.47% homology to that of SARS-CoV 11 , ACE2, which is identified as the crucial target for SARS-CoV, was also considered the host receptor of 2019-nCoV 12 . In fact, it was reported that host cells expressed ACE2, instead of APN and DPP4, were sensitive to 2019-nCoV 13, 14 . In vitro binding assay demonstrated that ACE2 enabled a potent interaction with 2019-nCoV S protein with an affinity of 14.7 nM 15 , which is 10-to 20-fold higher than that of ACE2 binding to SARS-CoV S protein.

In human, ACE2 is remarkably abundant on lung alveolar epithelial cells and small intestine enterocytes 16, 17 . As known, human intestinal epithelium encompasses approximately 200 m 2 of surface area 18 . Therefore, human intestine is conceivably susceptible to 2019-nCoV.

However, the occurrence of intestinal symptoms is lower than that of respiratory symptoms as a whole. In 38 patients infected by 2019-nCoV, only one diarrhea case was found 19 . Recent meta-analysis supported that digestive symptoms such as diarrhea, nausea, and vomiting were relatively rare compared with fever and cough 20 . These data indicate that intestinal epithelium is actually not so easy to be infected by 2019-nCoV as expected. The underlying reason is desirable to be explored.

It is well known that intestinal epithelium directly interacts with the external environment and encounters trillions of microorganisms including various viruses. To cope with the microbial threat, intestinal cells have evolved to produce a plenty of antimicrobial peptides (AMPs) to strengthen the mucosal barrier 21 . Defensin is a group of small (2-5 KD) and amphiphilic AMPs. Human defensins (HDs) are divided into two subgroups, α and β, based on the amino acid sequence homology and disulfide connectivity. Paneth cells, the major secretory cells located in the crypts of small intestine, specifically secrete two kinds of α defensins (HD5 and HD6) 22 . As reported, HD5 is the most abundant α defensin in intestine 23 .

Due to a lectin-like ability, HD5 can effectively bind lipids and glycosylated proteins 24,25 .

Because 2019-nCoV spike and ACE2 are both glycosylated proteins, there is a high possibility for HD5 to interact with 2019-nCoV spike or ACE2 or both of them and thereby disturb viral entry into host cells.

In the present study, we demonstrated that HD5 displayed a strong ability to prevent 2019-nCoV S1 to bind to intestinal epithelial cells by blocking the ligand-binding domain (LBD) of ACE2. Our findings unmasked the innate defense function of intestinal HD5 against 2019-nCoV, which may benefit the on-going development of preventive and therapeutic drugs.

Intestinal defensins are concentrated in the mucus layer after being secreted by Paneth cells and can contact with ACE2 receptor that locates on the brush border of the enterocytes 16, 21, 26 .

To gain an insight into the molecular interaction, we analyzed the recruitments of HD5 and HD6 to ACE2 by biolayer interferometry (BLI). The affinity of HD5 binding to recombinant human ACE2 immobilized on AR2G biosensors was 39.3 nM ( Figure 1A ), whereas no association signal was observed for HD6 ( Figure 1B) . The binding affinity of HD5 was much higher than that of a hexapeptide inhibitor, 438 YKYRYL 443 , derived from the RBD of SARS-CoV spike protein (46 μM) 27 . We previously discovered that HD5 is reduced to linear peptide, HD5 RED , under the catalysis of thioredoxin system in vivo 28, 29 . Interestingly, BLI revealed that similar to HD6, HD5 RED failed to bind ACE2 ( Figure 1C ). The structure-dependent interaction of HD5 with ACE2 is consistent with that cysteine replacement impairs the bactericidal and immunoregulatory efficiencies of HD5 30,31 , which expands the known roles of disulfide pairings in keeping the conformation of defensins.

The interaction between 2019-nCoV S1 and ACE2 was measured as well. The purified S1 expressed by human HEK293 cells intensively bound ACE2 with an affinity of 2.68 nM ( Figure 1D ). This assay conforms to the fact that virus forwardly contact ACE2 located on the cell surface and may avoid the steric hindrance of a receptor binding to its ligand. We reversely loaded S1 and measured the recruitment of ACE2. As shown in Figure S1 , the binding thickness was low, whereas the affinity (3.8 nM) was comparable to that of S1 binding to ACE2. Also, we noticed that the S1-ACE2 affinity was higher than those of S-ACE2 and RBD-ACE2 interactions 15, 32 . The discrepancy may be attributed to different proteins employed in different experiments.

HD5 is constitutively expressed in vivo and acts as a peptide "ranger" regulating the intestinal microbiota 33 . The storage volume of HD5 in Paneth cells has been estimated 90-450 mg/cm 2 of ileal surface area 22 . In ileal fluid, HD5 content is quantified to 6-30 µg/mL 34 . Such a large dose of HD5 in intestine provides a possibility that it may capture ACE2 before 2019-nCoV lands to enterocytes, although the affinity of S1-ACE2 interaction is higher than that of HD5 binding to ACE2. To evaluate the effect of HD5 coating on ACE2 interacting with S1, a blocking experiment was conducted by monitoring the bindings of S1 to ACE2 covered with different doses of HD5. As shown in Figure 1E , HD5 lowered the thickness of S1 binding to ACE2, indicative of a protective role of this peptide on viral adhesion. As HD5 is lectin-like peptide with multivalent binding ability 24 , we asked if HD5 could bind and block S1. The affinity of HD5 binding to S1 was 82.8 nM (Figure 2A ), as revealed by BLI.

Nevertheless, HD5 had less of an effect on ACE2-S1 interaction ( Figure 2B) , possibly due to a binding site distant from the RBD. Recent studies have discovered some antibodies and peptides with high affinities binding to the spike 32, 35 . These candidates are placed hopes on suppressing 2019-nCoV. Of note, our findings imply that the binding affinity alone is insufficient to predict the therapeutic potential of drugs.

X-ray crystal diffraction has resolved the detailed structure of 2019-nCoV RBD bound with ACE2 at 2.45 Å resolution, in which 20 residues of N-terminal ACE2 constitute the LBD 36 . To validate the inhibitive effect of HD5 on 2019-nCoV S1 binding to ACE2, we further conducted a cell experiment, in which human intestinal epithelium Caco-2 cells were exposed to 8 µg/mL of 2019-nCoV S1. Confocal microscopy observed that S1 largely adhered to the cell surface in the absence of HD5 (Figure 4 , Figure S3 ). Nevertheless, when cells were preincubated with 100 µg/mL of HD5, the recruitment of S1 was dramatically reduced.

Western blot supported that HD5 markedly protected Caco-2 from the adherence of S1

( Figure 5A ). The content of S1 binding to the cells preincuabted with HD5 was 3.4-fold lower than that binding to the cells without treatment ( Figure 5B) . Notably, the protection of HD5 was in a dose-dependent manner ( Figure 5C ). As shown in Figure 5D , HD5 could significantly decrease S1 binding to epithelial cells at concentrations as low as 10 µg/mL, which is within the range of HD5 concentration in intestine 22 . The protective effect of HD5 was also observed for human renal proximal tubular epithelial cells abundant in ACE2 ( Figure   S4 ) 16, 17 . However, S1 pretreated with HD5 was still efficient to contact Caco-2, as revealed by either immunoblotting or immunofluorescence. These data are in line with the results shown in Figure 1E and 2B, demonstrating that HD5 inhibits 2019-nCoV S1 adhering to host cells possibly by blocking ACE2 but not S1.

Collectively, we found for the first time that lectin-like intestinal defensin can protect host cells against 2019-nCoV entry. Paneth cell-secreted HD5 efficiently bound and blocked ACE2 which locates on the surface of intestinal epithelial cells, lowering the recruitment of 2019-nCoV S1 ( Figure 6 ). It is an interesting explanation to the clinical phenomenon that few intestinal symptoms are observed in patients infected by 2019-nCoV 19 . Due to the immature Paneth cells and deficiency in HD5 39 , it is might also be a potential reason for the infection of a newborn 30 h after birth 40 . Similarly, adult patients receiving small intestine transplantation or suffering from inflammatory bowel diseases such as the Crohn's disease, whose HD5 content are deficient in vivo 41, 42 , might be more susceptible to 2019-nCoV than the healthy.

More importantly, as there is a shortage of effective drugs to prevent and treat 2019-nCoV infection, we think that it may be a useful strategy to increase the content of HD5 in vivo by immunoregulation or exogenous supplement of HD5 as we recently described 43 .

Peptide synthesis. Peptides were prepared by Chinese Peptide Company (Hangzhou, Zhejiang Province, CHN) and purified with an Agilent (Beijing, CHN) 1260 HPLC system equipped with a Phenomenex/Luna C18 column (5 μm, 4.6×150 mm). The purities and molecule masses of peptides determined by reverse-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry, respectively, were shown in Table S1 . BLI-based blocking assay was conducted by monitoring the binding thickness of S1-ACE2 interaction with the interference of HD5. S1 and ACE2 were loaded on HIS1K and AR2G biosensors, respectively, at a concentration of 15 μg/mL. The immobilized ACE2 was incubated with 200 and 50 nM HD5 for 300 s at 30 o C. After a 300 s of disassociation, the signals of 400 nM S1 binding to ACE2 were recorded for another 300 s. Otherwise, the immobilized S1 was covered with 100 nM HD5 for 300 s. The bindings of 100 nM ACE2 to S1 were recorded for 300 s after peptide disassociation. 44 ) and the LBD of ACE2 (PDB: 6ACG 45 ) was performed on ZDOCK server 46 . The Gromacs 2020 software package 47 , AMBER99SB-ILDN force field, and TIP3P water model were applied for the simulation with a time step of 2 fs. Firstly, 1000-step minimization was carried out. Then, for fully relaxation, four 1-ns pre-equilibration simulation with restrained coordinates of the atoms belonging to the heavy atoms, main chain, backbone, and C-α, respectively, were performed step by step. Finally, each of five production simulation with isothermal-isobaric (NPT) ensemble at 1 atm and 298 K was performed for 20 ns. The binding free energy was calculated using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method with g_mmpbsa procedure for Gromacs 48, 49 . The binding free energy was calculated using 500 snapshots sampled every 10 ps from total 5 ns trajectory.

Immunofluorescence microscopy. Caco-2 and HK-2 obtained from the cell bank of Chinese Academy of Sciences (CAS, Shanghai) and cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Thermo Fisher Scientific, Shanghai, CHN) containing 10% foetal bovine serum (FBS, Gibco) were seeded into a 12-well plate with sterile glass slides at a density of 2×10 5 cells/well. Cells cultured overnight were washed with PBS and preincubated with 100 μg/mL of HD5 at 37 o C for 15 min, followed by an addition of 8 μg/mL of S1. Co-incubation was further performed at 4 o C for 1 h. After three times of wash with PBS, cells were fixed in 4% paraformaldehyde. A primary anti-spike rabbit monoclonal antibody (40150-R007, Sino Biological, 1:100) and a goat anti-rabbit secondary antibody (Alexa Fluor 488, Invitrogen, Thermo Fisher Scientific) were employed to stain 2019-nCoV S1. Nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, C1002, Beyotime, Shanghai, CHN). A Zeiss LSM 780 NLO confocal microscope was employed to observe the cells.

Western blot. Caco-2 cells were seeded into a 6-well plate at a density of 1×10 6 cells/well. Cells preincubated with different concentrations of HD5 (10, 50, and 100 μg/mL) at 37 o C for 15 min were exposed to 20 μg/mL of 2019-nCoV S1 containing His-tag at 4 o C for 1 h. After three times of wash with PBS, cells were collected and processed with RIPA lysis and extraction buffer (89900, Thermo Fisher Scientific). S1 content was measured by determining the band thickness as we previously described 50 . A primary anti-His-tag mouse monoclonal antibody (AF5060, Beyotime, 1:1000) and a goat anti-mouse secondary antibody (A0216, Beyotime, 1:2000) were employed to detect S1. β-actin determined by a mouse monoclonal antibody (AA128, Beyotime, 1:1000) was used as a reference. Binding kinetics for HD5 and S1 loaded on HIS1K biosensors. Fits of the data to a 1:1 binding model are shown with red dashes. (B) BLI-based S1 blocking assay. The binding signals of 100 nM ACE2 to S1 coated with 100 nM HD5 are recorded for 120 s. recruit S1 than those without treatment. A total of 25 μg of each protein sample is resolved by 10% SDS-PAGE. (B) Histogram presenting S1 levels obtained by gray scanning relative to the reference. **, P < 0.05. (C) Shown is the protein band of S1 binding to Caco-2 treated with increasing concentrations of HD5. (D) HD5 protects intestinal cells from the adherence of S1 in a dose dependent manner. **, P < 0.05, relative to the group of S1 without treatment. 

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

A Novel Coronavirus from Patients with Pneumonia in China

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical characteristics of 82 death cases with COVID-19. medRxiv

The spike protein of SARS-CoV -A target for vaccine and therapeutic development

From genome to antivirals: SARS as a test tube

Structural basis for the recognition of the 2019-nCoV by human ACE2. bioRxiv

Receptor recognition by novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS

Structure, Function, and Evolution of Coronavirus Spike Proteins

Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-nCoV

Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Multi-omics Evaluation of Gastrointestinal and Other Clinical Characteristics of SARS-CoV-2 and COVID-19

The gut microflora and intestinal epithelial cells: a continuing dialogue

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Incidence, clinical characteristics and prognostic factor of patients with COVID-19: a systematic review and meta-analysis. medRxiv

Epithelial antimicrobial defence of the skin and intestine

Paneth cell trypsin is the processing enzyme for human defensin-5

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii

Enteric defensins are essential regulators of intestinal microbial ecology

Crohn's disease: persistence in a complex with chymotrypsinogen and trypsin

The first-in-class peptide binder to the SARS-CoV-2 spike protein. bioRxiv

Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor. bioRxiv

Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor

Adaptation of SARS coronavirus to humans

Paneth Cells in Intestinal Health and Disease in the Newborn

Clinical characteristics of novel coronavirus disease 2019 (COVID-19) in newborns, infants and children

NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine

Inflammatory bowel disease

Succinylated casein-coated peptide-mesoporous silica nanoparticles as an antibiotic against intestinal bacterial infection

Crystal structures of human alpha-defensins HNP4, HD5, and HD6. Protein science : a publication of the

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2

ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers

GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers

A. g_mmpbsa-A GROMACS Tool for High-Throughput MM-PBSA Calculations

Electrostatics of nanosystems: application to microtubules and the ribosome

Klotho Protects Against Indoxyl Sulphate-Induced Myocardial Hypertrophy

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81725019 and 81703395), the program for scientific and technological innovation leader of Chongqing, the Natural Science Fund of Chongqing City (cstc2019jcyj-msxmX0011), and the frontier specific projects of Xinqiao Hospital (2018YQYLY004). We have no competing financial interests to declare.