Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions

Article abstract of DOI:10.1016/j.antiviral.2020.104873

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681–686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (K_D = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC₅₀ of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453–459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.

Full text of DOI:10.1016/j.antiviral.2020.104873

Journal Pre-proof
Characterization of heparin and severe acute respiratory syndrome-related
coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions
So Young Kim, Weihua Jin, Amika Sood, David W. Montgomery, Oliver C. Grant,
Mark M. Fuster, Li Fu, Jonathan S. Dordick, Robert J. Woods, Fuming Zhang, Robert
J. Linhardt
PII:
S0166-3542(20)30287-4
DOI:
https://doi.org/10.1016/j.antiviral.2020.104873
AVR 104873
Reference:
To appear in: Antiviral Research
Received Date: 6 May 2020
Revised Date: 30 June 2020
Accepted Date: 2 July 2020
Please cite this article as: Kim, S.Y., Jin, W., Sood, A., Montgomery, D.W., Grant, O.C., Fuster, M.M.,
Fu, L., Dordick, J.S., Woods, R.J., Zhang, F., Linhardt, R.J., Characterization of heparin and severe
acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions,
Antiviral Research, https://doi.org/10.1016/j.antiviral.2020.104873.
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Characterization of heparin and severe acute respiratory syndrome-
related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding
interactions
So Young Kima,b*, Weihua Jinf, Amika Soodd, David W. Montgomerye, Oliver C. Grante, Mark
M. Fustera,b,c, Li Fuf, Jonathan S. Dordickg, Robert J. Woodse, Fuming Zhangg*, Robert J.
Linhardtf,g,h,i*
a Department of Medicine, Division of Pulmonary and Critical Care, University of California San
Diego, La Jolla, California, United States of America
b VA San Diego Healthcare System, Medical and Research Sections, La Jolla, California, United
States of America
c
Glycobiology Research and Training Center, University of California San Diego, La Jolla,
California, United States of America
d
Department of Biostatistics and Bioinformatics, Duke University, Durham, North Carolina,
United States of America
e Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States
of America
f Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New
York, United States of America
g
Department of Chemical and Biological Engineering, Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of
America
h
Department of Biological Science, Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute, Troy, New York, United States of America
i Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York, United States of America
Prepared for Antiviral Research, May 2020
* Corresponding authors.
Email addresses: y0k001@health.ucsd.edu (S.Y. Kim), zhangf2@rpi.edu (F. Zhang), and
linhar@rpi.edu (R.J. Linhardt).
1

Abbreviations
ACE2, angiotensin-converting enzyme 2; CoV, coronavirus; COVID-19, coronavirus disease
2019; CSA, chondroitin sulfate A; CSC, chondroitin sulfate C; CSD, chondroitin sulfate D; CSE,
chondroitin sulfate E; dp, degree of polymerization; DS, dermatan sulfate; DPP4, dipeptidyl
peptidase 4; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HIV-1,
human immunodeficiency virus type 1; HS, heparan sulfate; HP, heparin; IBV, infectious
bronchitis virus; MERS-CoV, Middle East respiratory syndrome-related coronavirus; MHV,
mouse hepatitis virus; NACH, non-anticoagulant heparin; N-DeS HP, N-desulfated heparin; PG,
proteoglycans; RBD, receptor binding domain; RU, resonance unit; SPG, spike glycoprotein; S1,
subunit 1 of spike glycoprotein; S2, subunit 2 of spike glycoprotein; SARS-CoV, severe acute
respiratory syndrome-related coronavirus; SARS-CoV-2, severe acute respiratory syndrome-
related coronavirus 2; SDS, sodium dodecyl sulfonate; SPR, surface plasmon resonance;
TMPRSS2, transmembrane serine protease 2; TriS, trisulfated; 2-DeS HP, 2-O-desulfated
heparin; 6-DeS HP, 6-O-desulfated heparin derivative; 2OST/6OST, 2-/6-O-sulfotransferase
2

Abstract
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a
pandemic and continues to spread around the globe at an unprecedented rate. To date, no
effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a
novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site
(681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike
glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2
SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we
found that both monomeric and trimeric SARS-CoV-2 spike bind more tightly to immobilized
heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500
nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-
anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against
SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM,
respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate
interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric
SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding
domain is in an open conformation. The current study serves a foundation to further investigate
biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide
additional basis for further heparin-based interventions for COVID-19 patients exhibiting
thrombotic complications.
Keywords: SARS-CoV-2, COVID-19, glycosaminoglycans, heparin, spike glycoprotein,
binding interactions.
3

1. Introduction
In March 2020, the World Health Organization (WHO) declared severe acute respiratory
syndrome-related coronavirus 2 (SARS-CoV-2) a pandemic less than three months after its
initial emergence in Wuhan, China (Chan et al., 2020b; World Health Organization, 2020a).
SARS-CoV-2 is a zoonotic Betacoronavirus transmitted through person-person contact through
airborne and fecal-oral routes, and has caused over 10,021,401 confirmed coronavirus disease
2019 (COVID-19) cases and 499,913 associated deaths worldwide as of June 29th 2020 (Chan et
al., 2020b; van Doremalen et al., 2020; World Health Organization, 2020b; Yeo et al., 2020).
While there is limited understanding of SARS-CoV-2 pathogenesis, extensive studies have been
performed on how its closely related cousins, SARS-CoV and MERS-CoV (Middle East
respiratory syndrome-related coronavirus), invade host cell. Upon initially contacting the surface
of a host cell, SARS-CoV and MERS-CoV exploit host cell proteases to prime their surface
spike glycoproteins (SGPs) for fusion activation, which is achieved by receptor binding, low pH,
or both (Belouzard et al., 2009; Matsuyama et al., 2018). The receptor binding domain (RBD)
resides within subunit 1 (S1) while subunit 2 (S2) facilitates viral-host cell membrane fusion
(Belouzard et al., 2009). Activated SGP undergoes a conformational change followed by an
initiated fusion reaction with the host cell membrane (Belouzard et al., 2009). Endocytosed
virions are further processed by the endosomal protease cathepsin L in the late endosome (Huang
et al., 2006; Matsuyama et al., 2018). Both MERS-CoV and SARS-CoV require proteolytic
cleavage at their S2’ site, but not at their S1-S2 junction, for successful membrane fusion and
host cell entry (Belouzard et al., 2009; Matsuyama et al., 2018). Additionally, receptors involved
in fusion activation of SARS-CoV and MERS-CoV include heparan sulfate (HS) and
4

angiotensin-converting enzyme 2 (ACE2), and dipeptidyl peptidase 4 (DPP4), respectively (Lang
et al., 2011; Li et al., 2005; Raj et al., 2013). Recent progress confirms that SARS-CoV-2 also
utilizes ACE2 and host cell proteases during host cell entry (Hoffmann et al., 2020).
SARS-CoV and other pathogens arrive at a host cell surface by clinging, through their
surface proteins, to linear, sulfated polysaccharides called glycosaminoglycans (GAGs)
(Belouzard et al., 2012; Kamhi et al., 2013; Kim et al., 2017). The repeating disaccharide units of
GAGs, comprised of a hexosamine and a uronic acid or a galactose residue, are often sulfated
(Fig. S1) (Lindahl et al., 2015). GAGs are generally found covalently linked to core proteins as
proteoglycans (PGs) and reside inside the cell, at the cell surface, and in the extracellular matrix
(ECM) (Lindahl et al., 2015). GAGs facilitate various biological processes, including cellular
signaling, pathogenesis, and immunity, and possess diverse therapeutic applications (Lindahl et
al., 2015). For example, an FDA approved anticoagulant heparin (HP) is a secretory GAG
released from granules of mast cells during infection (Kim et al., 2018; Lindahl et al., 2015).
Some GAG binding proteins can be identified by amino acid sequences known as Cardin-
Weintraub motifs corresponding to ‘XBBXBX’ and ‘XBBBXXBX’, where X is a hydropathic
residue and B is a basic residue, such as arginine and lysine, responsible for interacting with the
sulfate groups present in GAGs (Cardin and Weintraub, 1989; Hileman et al., 1998a).
Examination of the SARS-CoV-2 SGP sequence revealed that the GAG-binding motif resides
within S1-S2 proteolytic cleavage motif (furin cleavage motif BBXBB) that is not present in
SARS-CoV or MERS-CoV SGPs (Fig. 1, S2, and S3) (Coutard et al., 2020). Additionally, we
discovered GAG-binding-like motifs within RBD and S2’ proteolytic cleavage site in SARS-
CoV-2 SGP (Fig. 1, S2, and S3). This discovery prompted us to hypothesize that host cell
surface GAGs may interact SARS-CoV-2 SGPs to potentially facilitate host cell entry. We
5

performed surface plasmon resonance (SPR)-based binding assays to determine binding kinetics
of the interactions between various GAGs and SARS-CoV-2 SGP in comparison with SARS-
CoV, and MERS-CoV SGP to address this question. Lastly, we performed blind docking on the
trimeric SARS-CoV-2 SGP model to objectively identify the preferred binding GAG-binding
sites on the SGP.
2. Materials and methods
2.1. Materials
Monomeric SARS-CoV-2, SARS-CoV, and MERS-CoV SGPs were purchased from
Sino Biological Inc. Trimeric SARS-CoV-2 SGP was kindly provided by Prof. Jason McLellan
from University of Texas at Austin (Wrapp et al., 2020). The GAGs used in this study were
porcine intestinal HP (HP) (average molecular weight, Mw = 16 kDa) and porcine intestinal
heparan sulfate (HS) (Mw = 14 kDa) from Celsus Laboratories (Cincinnati, OH); chondroitin
sulfate A (CSA, Mw = 20 kDa) from porcine rib cartilage (Sigma, St. Louis, MO), dermatan
sulfate (DS, Mw = 30 kDa) from porcine intestine (Sigma), chondroitin sulfate C (CSC, Mw =
20 kDa) from shark cartilage (Sigma), chondroitin sulfate D (CSD, Mw = 20 kDa) from whale
cartilage (Seikagaku, Tokyo, Japan) and chondroitin sulfate E (CSE, Mw = 20 kDa) from squid
cartilage (Seikagaku). N-desulfated HP (N-DeS HP, Mw = 14 kDa) and 2-O-desulfated HP (2-
DeS HP, MW = 13 kDa) were prepared in house based on previously established protocols
(Yates et al., 1996). The 6-O-desulfated HP derivative, 6-DeS HP, Mw = 13 kDa, was
generously provided by Prof. Lianchun Wang from University of South Florida. Non-
6

anticoagulant low molecular weight HP (NACH) was synthesized from dalteparin, a nitrous acid
depolymerization product of porcine intestinal HP, followed by periodate oxidation as described
in our previous work (Islam et al., 2002). TriS HS (NS2S6S) was synthesized from N-sulfo
heparosan with subsequent modification with C5-epimerase and 2-O- and 6-O-sulfotransferases
(2OST and 6OST1/6OST3) (Linhardt et al., 2007). HP oligosaccharides included tetrasaccharide
(dp4), hexasaccharide (dp6), octasaccharide (dp8), decasaccharide (dp10), dodecasaccharide
(dp12), tetradecasaccharide (dp14), hexadecasaccharide (dp16) and octadecasaccharide (dp18)
and were prepared from porcine intestinal HP controlled partial heparin lyase 1 treatment
followed by size fractionation. The chemical structures of the GAGs are shown in Fig. S1. The
SA sensor chips were from GE Healthcare (Uppsala, Sweden).
SPR measurements were
performed on a BIAcore 3000 operated using BIAcore 3000 control and BIAevaluation software
(version 4.0.1).
2.2. Preparation of HP biochip
Biotinylated HP was prepared by conjugating its reducing end to amine-PEG3-Biotin
(Pierce, Rockford, IL). In brief, HP (2 mg) and amine-PEG3-Biotin (2 mg, Pierce, Rockford, IL)
were dissolved in 200 µl H2O, 10 mg NaCNBH3 was added. The reaction mixture was heated at
70 °C for 24 h, after that a further 10 mg NaCNBH3 was added and the reaction was heated at
70 °C for another 24 h. After cooling to room temperature, the mixture was desalted with the
spin column (3,000 MWCO). Biotinylated HP was collected, freeze-dried and used for SA chip
preparation. The biotinylated HP was immobilized to streptavidin (SA) chip based on the
manufacturer’s protocol. The successful immobilization of HP was confirmed by the
observation of a 200-resonance unit (RU) increase on the sensor chip. The control flow cell
7

(FC1) was prepared by 2 min injection with saturated biotin.
2.3. Measurement of interaction between HP and CoV SGP using BIAcore
SGP samples were diluted in buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005%
surfactant P20, pH 7.4,). Different dilutions of protein samples were injected at a flow rate of 30
µL/min. At the end of the sample injection, the same buffer was flowed over the sensor surface
to facilitate dissociation. After a 3 min dissociation time, the sensor surface was regenerated by
injecting with 30 µL of 0.25% sodium dodecyl sulfonate (SDS) to get fully regenerated surface.
The response was monitored as a function of time (sensorgram) at 25 °C.
2.4. Solution competition study between HP on chip surface and HP, HP-derived
oligosaccharides, chemically modified HP or GAGs in solution using SPR
SARS-CoV-2 SGP (50 nM) mixed with 1 µM HP, HP-derived oligosaccharides,
chemically modified HP or GAGs in SPR buffer were injected over HP chip at a flow rate of 30
µL/min, respectively. After each run, the dissociation and the regeneration were performed as
described above.
2.5. SPR solution competition IC50 measurement of glycans (HP, TriS HS and NACH) inhibition
on SARS-CoV-2 SGP-HP interaction
Solution competition studies between a surface HP and soluble glycans (HP, TriS HS and
NACH) to measure IC50 were performed using SPR (Fu et al., 2013). In brief, SARS-CoV-2 S-
protein (50 nM) samples alone or mixed with different concentrations of glycans in SPR buffer
were injected over the HP chip at a flow rate of 30 µl/min, respectively. After each run,
8

dissociation and regeneration were performed as described above. For each set of competition
experiments, a control experiment (only protein without glycan) was performed to ensure the
surface was completely regenerated.
2.6. Protein modeling
The 3D coordinates for the SGP trimer (NCBI reference sequence YP_009724390.1)
were downloaded from the SWISS-MODEL homology modeling server (Waterhouse et al.,
2018). The selected model was generated with the Cryo-EM structure PDB ID 6VSB as a
template, which has a 99.26% sequence identity and 95% coverage for amino acids 27 to 1146.
The template and resulting model is the “prefusion” structure with one of the three receptor
binding domains (Chain A) in the “up” or “open” conformation (Wrapp et al., 2020). Cryo-
EM studies have revealed that the SARS-CoV-2 SGP trimer exists in two conformational states
in approximately equal abundance (Walls et al., 2020). In one state, all SGP monomers have
their hACE2-binding domain closed, and in the other, one monomer has its hACE2-binding
domain open, where it is positioned away from the interior of the protein.
2.7. Ligand docking
Initial coordinates for a hexasaccharide fragment of HS (GlcA(2S)-GlcNS(6S))3 were
generated using the GAG-Builder tool (Singh et al., 2019) at GLYCAM-Web (glycam.org) and
used for unbiased (blind) docking. A hexasaccharide was chosen as being sufficiently long to
represent a typical GAG length found in protein co-complexes (Singh et al., 2019) and to avoid
introducing so many degrees of internal flexibility that the efficiency of the docking
conformational search algorithm was impaired. Docking was performed using a version of Vina-
9

Carb (Nivedha et al., 2016) that has been modified to improve its performance for GAGs. A grid
box with dimensions (x = 190, y = 223, z = 184 Å) was placed at the geometric center the protein
enclosing its entire surface. Docking was performed with default values, with the following
exceptions: exhaustiveness = 80, chi_cutoff = 2, and chi_coeff = 0.5. All sulfate and hydroxyl
groups and glycosidic torsion angles were treated as flexible, resulting in 83 ligand poses.
3. Results
3.1. Kinetic measurements of CoV SPG-HP interactions
Previous reports showed that various CoVs bind GAGs through their SGPs to invade host
cells (Belouzard et al., 2012). In the current study, we utilized SPR to measure the binding
kinetics and interaction affinity of monomeric and trimeric SARS-CoV-2, monomeric SARS-
CoV and MERS-CoV with SGP-HP using a sensor chip with immobilized HP. Sensorgrams of
CoV SGP-HP interactions are shown in Fig. 2. The sensorgrams were fit globally to obtain
association rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant
(KD) (Table 1) using the BiaEvaluation software and assuming a 1:1 Langmuir model. SARS-
CoV-2 and MERS CoV SGP exhibited a markedly low dissociation rate constant (kd ~ 10-7 1/s)
suggesting excellent binding strength. The HP binding properties of monomeric SARS-CoV-2
SGP were comparable to that of the trimeric form (KD of monomer and trimer were 40 pM and
73 pM, respectively). In comparison, previously known HP binding SARS-CoV SGP showed
nearly 10-fold lower affinity, 500 nM. The length of monomeric SARS-CoV-2 SGP (Sino
Biological Inc.) spans subunit 1 containing proposed GAG-binding motifs 1 and 2 while trimeric
SARS-CoV-2 SGP was expressed and characterized by Prof. Jason McLellan (Wrapp et al.,
10

2020). Comparable KD values suggest that the folding of monomeric SGP is in the same correct
conformation as the trimer. Trimeric SPG, surprisingly, does not possess greater binding affinity
than that of monomer, this may be partly due to the monomer having both GAG-binding motif
sites 1 and 2 while trimer has three of the GAG-binding site 1 and mutated site 2 from RRAR to
GSAS (Wrapp et al., 2020).
The extremely high binding affinity of SARS-CoV-2 SGP to HP was supported by the
chip surface regeneration conditions. The immobilized HP surface could only be regenerated
using a harsh regeneration reagent, 0.25% SDS, instead of the standard 2M NaCl solution used
for removing HP-binding proteins. One reason for SARS-CoV-2 SGP’s extremely high affinity
to immobilized heparin is the high density of surface bound ligands might promote polyvalent
interactions. The difference of binding kinetics and affinity of CoV SGPs to HP may also be due
in part to the difference in protein sequence of the CoV SGPs. Based on amino acid alignment
analysis using the Basic Local Alignment Search Tool (BLAST), SARS-CoV and SARS-CoV-2
SGPs share 76% similarity. Association rate constants (ka) for MERS-CoV SGP (339 (± 27)
1/M-1s1) was the lowest, followed by monomeric and trimeric SARS-CoV-2 SGPs (2.5 × 103 (±
62.7) M-1s-1 and 1.6 × 103 (± 127) M-1s-1, respectively) (Table 1). SARS-CoV SGP had the
highest Ka, which was 4.12 × 104 (± 136) M-1s-1. The differences in ka values suggest a different
mechanism when each SGP binds HP in addition to differences in binding strengths.
3.2. SPR solution competition study on the interaction between surface-bound HP and SARS-
CoV-2 SGP to HP-derived oligosaccharides in solution
Solution/surface competition experiments were performed by SPR to examine the effect
of the saccharide chain length of HP on the SARS-CoV-2 SGP-HP interaction. HP-derived
11

oligosaccharides of different lengths, from tetrasaccharide (dp4) to octadecasaccharide (dp18),
were used in these competition studies. The same concentration (1000 nM) of HP
oligosaccharides were mixed in the SARS-CoV-2 SGP protein (50 nM)/ HP interaction solution.
Negligible competition was observed (Fig. S4) when 1000 nM of oligosaccharides (from dp4 to
dp18) were present in the protein solution suggesting that the SARS-CoV-2 SGP-HP interaction
is chain-length dependent and it prefers to bind full chain (~dp30) HP.
3.3. SPR solution competition study of different chemically modified HP derivatives and GAGs
Competition levels measured by SPR for chemically modified HP derivatives are shown
in Fig. 3. The results of these studies demonstrate that all the chemically modified HPs, N-
desulfated HP, 2-O-desulfated HP and 6-O-desulfated HP, were unable to compete with
immobilized HP for binding to SARS-CoV-2 SGP-HP suggesting all the sulfate groups within
HP have critical impact on this interaction.
SPR competition assay was also used to test the binding preference of SARS-CoV-2 SGP
for various GAGs (Fig. S1), including various chondroitin sulfates, dermatan and keratan
sulfates, and the results are shown in Fig. S5. Weak or no inhibitory activities were observed for
all GAGs tested, suggesting that the binding of SARS-CoV-2 SGP protein to GAGs appears to
be HP specific and greatly influenced by the level of sulfation within the GAG.
3.4. SPR solution competition dose response analysis of HP, Tri-sulfated HS and NACH
Solution competition dose response analysis between surface immobilized HP and
various soluble glycans (HP, non-anticoagulant trisulfated (TriS) HS, and non-anticoagulant low
molecular weight HP (NACH)) was performed to calculate their IC50 values (Fig. 4A-4E).
SARS-CoV-2 SGP protein (50 nM) samples were pre-mixed with different concentrations of
12

glycans before injection into the HP chip. The sensorgrams (Fig. 4A, 4C, and 4E) show that
once the active binding sites on SARS-CoV-2 SGP were occupied by glycans in solution, the
binding of SARS-CoV-2 SGP to the surface-immobilized HP decreased resulting in a reduction
of signal in a concentration dependent fashion. The IC50 values (concentration of competing
analyte resulting in a 50% decrease in RU) were calculated from the plots SARS-CoV-2 SGP
binding signal (normalized) vs. glycans concentration in solution (Fig. 4B, 4D, and 4F). The
IC50 values of HP, TriS HS and NACH were 0.056 µM, 012 µM and 26.4 µM, respectively.
3.5. Identification of GAG-binding motifs by blinding docking analysis
Using a modified version of Autodock Vina tuned for use with carbohydrates (Vina-Carb)
(Nivedha et al., 2016; Trott and Olson, 2009), we performed blind docking on the trimeric
SARS-CoV-2 SGP model to discover objectively the preferred binding GAG-binding sites on the
SGP protein surface. The SGP contains three putative GAG-binding motifs with the following
sequences: 453-459 (YRLFRKS), 681-686 (PRRARS), and 810-816 (SKPSKRS), which we
define as sites 1, 2, and 3, respectively (Fig. 1, S2, and S3). An HS hexasaccharide fragment
(GlcA(2S)-GlcNS(6S)) binds site 2 in each monomer chain in the trimeric SGP (Fig. 5C and S3).
The docking results also indicates that HS may bind to site 1 when the apex of the S1 monomer
is in an open conformation, as this allows basic residues to be more accessible to ligand binding.
The site 1 residues are less accessible for GAG binding when the domain is in a closed
conformation (Fig. 5D). The electrostatic potential surface representation of the trimeric SGP
confirms that the GAG-binding poses generally prefer regions of positive charge, as expected,
and illustrates that basic residues within site 3 are not exposed for binding to HS on any of the
13

chains (Fig. 5A). Finally, our blind docking analysis reveals that a longer HS polymer may span
an inter-domain channel that contains site 2.
4. Discussion
The original SARS-CoV and numerous pathogens exploit host cell surface GAGs during
the initial step of host cell entry (Agelidis and Shukla, 2020; Belouzard et al., 2012; Kamhi et al.,
2013; Kim et al., 2017). Based on our initial discovery of GAG-binding and GAG-binding-like
motifs at site 1 (within the RBD, Y453-S459), site 2 (at the proteolytic cleavage site at S1/S2
junction, P681-S686), and site 3 (at the S2’ proteolytic cleavage site, S810-S816), we
hypothesized that SARS-CoV-2 may also interact with host cell surface GAGs through its SGPs
to invade host cell (Fig. 1, S2, and S3). The predominant GAG in normal human lung is HS
followed by CS (Frevert and Sannes, 2005) and it is noteworthy that lung tissue is rich in mast
cells and has been a source of commercial HP (Guan et al., 2016). Using unbiased docking, we
found that TriS HS hexasaccharide (GlcA(2S)-GlcNS(6S)) binds site 2 in each monomer chain
in the trimeric SARS-CoV-2 SGP (Fig. 5C). TriS HS hexasaccharide may additionally bind site
1 when the SGP monomer is in an open conformation, but not site 3 where the basic residues are
not accessible to the surface (Fig. 5C and 5D). Docking results indicated that the HS
hexasaccharides could span an inter-domain channel that includes site 2, suggesting a
mechanism for the binding of a longer HS sequence (Fig. 5C).
Next, we experimentally determined binding kinetics for the interactions between HP
(rich (60-80%) in TriS domains) and monomeric SARS-CoV-2, trimeric SARS-CoV-2,
14

monomeric SARS-CoV, and monomeric MERS-CoV SGPs using SPR binding assays (Fig. 2
and Table 1). GAG-protein interactions are mainly electrostatically driven (Hileman et al.,
1998b), thus, HS-binding proteins generally bind HP due to its higher degree of sulfation
(Lindahl et al., 2015). We discovered that HP binds both monomeric and trimeric SARS-CoV-2
SGP with remarkable affinity (KD = 40 pM and 73 pM, respectively) (Fig. 2 and Table 1). This
was unexpectedly tight binding for a GAG-protein interaction as even one of one of the
prototypical HP-binding proteins, fibroblast growth factor 2 (FGF2), has a KD of 39 nM
(Ibrahimi et al., 2004). Additionally, high affinity may be partly due to polyvalent interactions
that the high density of surface bound HP provides. In comparison, SARS-Cov and MERS-CoV
SGPs also bind HP, however, much more weakly with binding strengths of KD = 500 nM and 1
nM, respectively (Fig. 2 and Table 1). While HS facilitates SARS-CoV host cell entry and is an
essential host cell surface receptor, its involvement in MERS-CoV host cell entry or binding
kinetics for SARS-Cov and MERS-CoV SGPs had not previously been reported (Lang et al.,
2011).
After discovering the high binding affinity between HP and SARS-CoV-2 SGP, we next
found that the degree and position of sulfation within HP was important for its successful
binding to monomeric SARS-CoV-2 SGP (Fig. 3 and 4). N-, 2-O, and 6-O-sulfation were all
required for binding to SARS-CoV-2 SGP (Fig. 3). This was additionally demonstrated when
competitive binding studies gave IC50 values of HP (0.056 μM), TriS HS (NS2S6S) (0.12 μM),
and NACH (26.4 μM) for the inhibition of SARS-CoV-2 SGP binding to immobilized HP (Fig.
4). Both HP and TriS HS are sulfated at N-, 2-O-, and 6-O- positions and have approximately the
same molecular weight (and chain length) but HP has additional 3-O-sulfation, responsible for its
anticoagulant activity (Fig. S1). NACH lacks an intact antithrombin binding site, has a lower
15

average molecular weight of 5 kDa than HP, and a higher content (>90%) of TriS (Islam et al.,
2002).
The low IC50 of these GAGs suggest that the FDA approved anticoagulant HP, or its non-
anticoagulant derivatives, might have therapeutic potential against SARS-CoV-2 infection as
competitive inhibitors. The location of proposed GAG-binding sites is also of interest. Unlike
SARS-CoV and MERS-CoV SGPs, SARS-CoV-2 SGP has a novel insert in the amino acid
sequence (681-686 (PRRARS)) that fully follows GAG-binding Cardin-Weintraub motif
(XBBXBX) and a furin-cleavage motif (BBXBB) at the S1/S2 junction (Fig. 1). This site was
also shown to be a preferred GAG-binding motif by our unbiased docking study (Fig. 5).
Proteolytic cleavage at S1/S2 is not required for successful viral-host cellular membrane fusion
in SARS-CoV and MERS-CoV SGPs (Belouzard et al., 2009; Matsuyama et al., 2018).
Transmembrane serine protease 2 (TMPRSS2) and cathepsin B/L have been recently confirmed
as host cell proteases that SARS-CoV-2 exploits to prime for fusion activation and membrane
fusion (Hoffmann et al., 2020). Proteolytic cleavage primes the SGP for fusion activation and
may additionally influence cell-cell fusion, host cell entry, and/or the infectivity of the virus
(Belouzard et al., 2012; Follis et al., 2006). Additionally, it is notable that the proteolytic
cleavage motif PRRARS is almost identical to the HP binding sequence in fibronectin, PRRARV.
The nucleocapsid protein of SARS-CoV induces apoptosis and actin reorganization partly
through downregulation of fibronectin in mammalian cells under stressed conditions (Surjit et al.,
2004). It also has been proposed that SARS‐COV‐2 SGP has acquired the general binding motif
for integrin, one of fibronectin binding proteins for ECM maintenance, which facilitates virus
transmission efficiency (Tresoldi et al., 2020).
16

Some CoVs possess both GAG-binding and furin cleavage motifs at their S1/S2 junction
in their SGPs (Belouzard et al., 2012). In the case of MHV A59 SGP, a single amino acid
mutation near the GAG-binding and furin cleavage motifs, resulted from a cell culture adaptation
(known as MHV/BHK), determines whether a virion binds GAGs or exploits host cell surface
protease, but not both (de Haan et al., 2008, 2005). While not within the CoV family, human
immunodeficiency virus type 1 (HIV-1) requires HS-binding to achieve optimal furin processing
because HS binding allows selective exposure of furin cleavage site on the envelope
glycoprotein (Pasquato et al., 2007). It is of interest to further investigate potential crosstalk
between host cell surface GAGs and cellular proteases during SARS-CoV-2 host cell entry.
COVID-19 hospitalized patients appear to be at increased risk of thrombotic
complications and WHO recommends HP to reduce incidence of venous thromboembolism
(Giannis et al., 2020; World Health Organization, 2020c). Additional studies report that soluble
unfractionated heparin and its derivatives inhibit SARS-CoV-2 host cell entry in vitro (Partridge
et al., 2020; Tandon et al., 2020). Nebulized Heparin is currently being evaluated in COVID-19
patients (Dixon et al., 2020) and a separate clinical study demonstrates the association between
heparin and lower mortality in COVID-19 patients (Ayerbe et al., 2020). With additional
randomized controlled trials in various therapeutic regimes (Ayerbe et al., 2020), HP may be an
excellent candidate to be repurposed as prophylactic COVID-19 therapeutic.
Based on our findings, we propose a model on how GAGs may facilitate host cell entry
of SARS-CoV-2 (Fig. 6). First, virions land on the epithelial surface in the airway by binding to
HS through their SGPs (Fig. 6A). Host cell surface proteoglycans utilize their long HS chains to
securely wrap around the trimeric SGP (Fig. 6A). During this step, heavily sulfated HS chains
span inter-domain channel containing GAG-binding site 2 on each monomer in the trimeric SGP
17

and binds site 1 within the RBD in an open conformation (Fig. 5). Host cell surface and
extracellular proteases, including TMPRSS2 (Hoffmann et al., 2020), may process site 2 (S1/S2
junction) and/or 3 (S2’) and GAG chains come off from site 2 upon cleavage (Fig. 6B). HS and
an established host cell surface receptor ACE2 binding to more readily accessible RBD
containing site 1 may drive conformational change of SGP and activate viral-cellular membrane
fusion (Wrapp et al., 2020). A separate study reports that HP binding results in conformational
change in RBD demonstrated by circular dichroism (Mycroft-west et al., 2020). Finally, SGP on
the endocytosed virion may utilize an endosomal host cell protease, such as cathepsin B/L, to
further execute viral-cellular membrane fusion (Hoffmann et al., 2020) (Fig. 6C). If taken an
additional route of viral entry, receptor-dependent endocytosis, reported for SARS-CoV (Wang
et al., 2008), concurrently endocytosed and recycled HSPGs may be further exploited by SARS-
CoV-2.
In conclusion, we have characterized binding kinetics between GAGs and SARS-CoV-2
SGP and determined potential GAG-binding motifs within SARS-CoV-2 SGP in the current
work. SPR studies demonstrate that both monomeric and trimeric SARS-CoV-2 SGP bind HP
with remarkably high affinity and it prefers long, heavily sulfated (TriS rich) structures.
Additionally, we reported low IC50 of HP and derivatives against HP and SARS-CoV-2 SGP
interactions suggesting therapeutic potential of HP as COVID-19 competitive inhibitors. Lastly,
unbiased computational ligand docking indicated that a TriS HS oligosaccharide preferably
interacts with GAG-binding motifs at the S1/S2 junction and within receptor binding domain and
hinted at mechanism of binding. This study provides groundwork for biological evaluation of
GAGs as host cell surface receptors and facilitate designing of GAG-based COVID-19
therapeutics.
18

Acknowledgements
We appreciate Prof. Jason McLellan from University of Texas Austin for providing trimeric
SARS-CoV-2 SGP. Additionally, we thank professor Lianchun Wang from University of South
Florida for providing 6-O-desulfated HP derivative. This work was supported by the National
Heart, Lung, and Blood Institute Grant K12 5K12HL141956-02 to S.Y.K. and M.M.F.; the
National Institutes of Health Grants U01 CA221216 and U01 CA207824 to R.J.W.; and the
National Institutes of Health Grants DK111958, CA231074, NS088496, and AG062344 to R.J.L.
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26

Table 1 Summary of kinetic data of CoV SGP-HP interactions*
Interaction
ka (M-1s1)
kd (1/s)
KD (M)
2.5 × 103
(±62.7)
1.6 × 103
(±127)
4.12 × 104
(±136)
339
1.0 × 10-7
(±7.9 × 10-8)
1.2 × 10-7
4.0 × 10-11
SARS-CoV-2 SGP
(monomer)
7.3 × 10-11
5.0 × 10-7
1.0 × 10-9
SARS-CoV-2 SGP
(trimer)
(±5.5 × 10-8)
4.01 × 10-4
(±6.49 × 10-6)
3.5 × 10-7
SARS-CoV SGP
(monomer)
MERS-CoV SGP
(monomer)
(±2.6 × 10-6)
(±27)
*The data with (±) in parentheses are the standard deviations (SD) from global fitting of five
injections.
27

Figure legends
Fig. 1. Identification of GAG-binding motif within SARS-CoV-2, SARS-CoV, and MERS-CoV
SGPs. Domains in SGP include signal peptide (SP), N-terminal domain (NTD), receptor-binding
domain (RBD), fusion peptide (FP), heptad repeat 1/2 (HR 1/2).
Fig. 2. SPR sensorgrams for binding kinetics/affinity measurements for SGP-HP interactions.
(A) SARS-CoV-2 SGP (monomer), concentration of SGP (from top to bottom): 100, 50, 25, 12.5
and 6.25 nM. (B) SARS-CoV SGP, concentrations of SARS-CoV SGP (from top to bottom):
100, 50, 25, 12.5 and 6.25 nM. (C) MERS CoV SGP, concentrations of MERS CoV SGP (from
top to bottom): 100, 50, 25, 12.5 and 6.25 nM. (D) SARS-CoV-2 SGP (trimer), concentration of
SGP (from top to bottom): 800, 400, 200, 100 and 50 nM. The black curves are the fits using a
1:1 Langmuir model from BIAevaluate 4.0.1.
Fig. 3. Bar graphs of normalized SARS-CoV-2 SGP binding preference to surface HP by
competing with different chemical modified HP in solution. Concentration was 50 nM for
SARS-CoV-2 SGP and 1000 nM for different chemical modified HP. All bar graphs based on
triplicate experiments.
Fig. 4. Inhibition analysis of glycans on the interactions between SARS-CoV-2 SGP and HP
using SPR; SARS-CoV-2 SGP concentration was 50 nM. (A) Competition SPR sensorgrams of
SARS-CoV-2 SGP-HP interaction inhibiting by different concentration of heparin. (B) Dose
response curves for IC50 calculation of heparin using SARS-CoV-2 SGP inhibition data from
surface competition SPR. (C) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP
28

interaction inhibiting by different concentration of TriS HS. (D) Dose response curves for IC50
calculation of TriS HS using SARS-CoV-2 SGP inhibition data from surface competition SPR.
(E) Competition SPR sensorgrams of SARS-CoV-2 SGP-HP interaction inhibiting by different
concentration of NACH. (F) Dose response curves for IC50 calculation of NACH using SARS-
CoV-2 SGP inhibition data from surface competition SPR.
Fig. 5. Structure of trimeric SARS-CoV-2 SGP and proposed GAG-binding motifs. (A)
Electrostatic potential surface (-ve charge (red) to +ve charge (blue)) computed with Chimera. (B)
Electrostatic potential surface showing a top view of the SGP trimer. (C) Solvent accessible
surface of the SARS-CoV-2 SGP trimer (pink (Chain A), grey (Chain B), blue (Chain C))
showing the predicted poses of HS hexasaccharides (orange) obtained from unbiased docking,
and the three GAG-binding motifs (yellow (Chain A), white (Chain B), red (Chain C)), image
generated with VMD (Humphrey et al., 1996). (D) Solvent accessible surface showing a top
view of the SGP trimer. Amino acid sequences for GAG-binding motifs site 1, 2, and 3 are
YRLFRKS, PRRARS, and SKPSKRS.
Fig. 6. Proposed model of SARS-CoV-2 host cell entry. SARS-CoV-2 surface is decorated with
envelop (E), membrane (M), and SGP (Chan et al., 2020a). (A) Virion lands on host cell surface
by binding to heparan sulfate proteoglycan (HSPG). (B) SGP goes through proteolytic digestion
by host cell surface protease, which initiates viral-host cell membrane fusion by conformational
change caused by host cell receptor binding (HSPG and ACE2). ACE2 is an established host cell
surface receptor in SARS-CoV-2 host cell entry (Hoffmann et al., 2020; Wrapp et al., 2020). (C)
Virion enters the host cell and may further experience proteolytic processing by endosomal host
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