SARS-CoV-2 spike protein binds heparan sulfate in a length- and sequence-dependent manner

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. Hexa- and octasaccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with higher affinity to heparin (KD 55 nM) compared to the receptor binding domain (RBD, KD 1 µM) alone. An octasaccharide composed of IdoA2S-GlcNS6S could inhibit spike-heparin interaction with an IC50 of 38 nM. Our data supports a model in which the RBD of the spike of SARS-CoV-2 confers sequence specificity for HS expressed by target cells whereas an additional HS binding site in the S1/S2 proteolytic cleavage site enhances the avidity of binding. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.


INTRODUCTION
The SARS-CoV-2 pandemic demands the urgent development of therapeutic strategies. An attractive approach is to interfere in the attachment of the virus to the host cell. The entry of SARS-CoV-2 into cells is initiated by binding of the transmembrane spike (S) glycoprotein of the virus to angiotensin-converting enzyme 2 (ACE2) of the host. 1 SARS-CoV is closely related to SARS-CoV-2 and employs the same receptor. 2 The spike protein of SARS-CoV-2 is comprised of two subunits; S1 is responsible for binding to the host receptor, whereas S2 promotes membrane fusion. The C terminal domain (CTD) of S1 harbors the receptor binding domain (RBD). 3 It is known that the spike protein of number of human coronaviruses can bind to a secondary receptor, or co-receptor, to facilitate cell entry. For example, MERS-CoV employs sialic acid as co-receptor along its main receptor DPP4. 4 Human CoV-NL63, which also utilizes ACE2 as receptor, uses heparan sulfate (HS) proteoglycans, as coreceptor. 5 It has also been shown that entry of SARS-CoV pseudo-typed virus into Vero E6 and Caco-2 cells can significantly be inhibited by heparin or treatment with heparinase, indicating the importance of HS for infectivity. 6 There are indications that the SARS-CoV-2 spike also interacts with HS. One reports showed that heparin can induce a conformation change in the RBD of SARS-CoV-2. 7 A combined SPR and computational study showed that glycosaminoglycans can bind to the proteolytic cleavage site of the S1 and S2 protein. 8 HS are highly complex O-and N-sulfated polysaccharides that reside as major components on the cell surface and extracellular matrix of all eukaryotic cells. 9 Various proteins interact with HS thereby regulating many biological and disease processes, including cell adhesion, proliferation, differentiation and inflammation. They are also used by many viruses, including herpes simplex virus (HSV), Dengue virus, HIV, and various coronaviruses, as receptor or co-receptor. [10][11][12] The biosynthesis of HS is highly regulated and the length and degree and pattern of sulfation of HS can differ substantially between different cell types. The so-called "HS sulfate code hypothesis" is based on the notion that expression of specific HS epitopes by cells makes it possible to recruit specific HS-binding proteins, thereby controlling a multitude of biological processes. [13][14] In support of this hypothesis, several studies have shown that HS binding proteins exhibit preferences for specific HS oligosaccharide motifs. [15][16] Therefore, we were compelled to investigate whether the spike of SARS-CoV-2 recognizes specific HS motifs. Such insight is expected to pave the way to develop inhibitors of viral cell binding and entry.
Previously, we prepared an unprecedented library of structurally well-defined heparan sulfate oligosaccharides that differ in chain length, backbone composition an sulfation pattern. [17][18] This collection of HS oligosaccharides was used to develop a glycan microarray for the systematic analysis of binding selectivities of HS-binding proteins.
Using this microarray platform in conjugation with detailed binding studies, we found that the SARS-CoV-2-S can bind HS in a length-and sequence-dependent manner. We propose a model for HS mediated cell binding and entry.

RESULTS AND DISCUSSION
A HS microarray was fabricated having well over 100 unique di-, tetra-hexa-and octasaccharides differing in backbone composition and sulfation pattern (Fig. 1C). The synthetic HS oligosaccharides contain an aminopentyl spacer allowing printing on Nhydroxysuccinimide (NHS)-active glass slides for microarray fabrication. All oligosaccharides were printed at 100 µM concentration in replicates of 6 by non-contact piezoelectric printing. The quality of the HS microarray was validated using various well characterized HS-binding proteins.
The spike glycoprotein of SARS-CoV-2 (S1+S2, extra cellular domain, amino acid residue 1-1213) was expressed in insect cells having a C-terminal His-tag. 19 Recombinant SARS-CoV-2-RBD, containing amino acid residue 319-541, was expressed in HEK293 cells also with a C-terminal His-tag. 19 Sub-arrays were incubated with different concentrations of the proteins in a binding buffer (pH 7.4, 20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 with 1% BSA and 0.05% Tween-20) at room temperature for 1 h. After washing and drying, the subarrays were exposed to an anti-His antibody labeled with AlexaFluor® 647 for another hour.
Binding was established by fluorescent scanning and the resulting data was processed using home-written software.
To analyze the data, the compounds were arranged according to increasing backbone length, and within each group with increasing numbers of sulfates. Intriguingly, the proteins showed a strong preference for specific HS oligosaccharides (Fig. 1A, B).
Furthermore, it was found that SARS-CoV-2-spike and the RBD have similar selectivities. the RBD, another HS-binding site may reside in the S1/S2 proteolytic cleavage site of the spike. 8 Our data support a model in which the HS binding site of the RBD confers sequence specificity whereas that in S1/S2 proteolytic cleavage site enhances the affinity.
Next, we examined whether HS oligosaccharide 81 can interfere in the interaction of the spike or RBD with immobilized heparin. Thus, the spike protein (150 nM) or RBD (2.4 µM) were pre-mixed with different concentrations of compound 81 and then used as analytes. The IC50 values were determined by non-linear fitting of Log(inhibitor) vs. response using variable slope (Fig. 3). The IC50 values for the spike protein and RBD are 38 nM and 264 nM, respectively, showing compound 81 is a potent competitive inhibitor of the interaction of SARS-CoV-2 spike with heparin.

DISCUSSION AND CONCLUSIONS
The microarray and SPR results presented here demonstrate that the spike of SARS-CoV-2 can bind HS in a length-and sequence-dependent manner. Hexa-and octasaccharides composed of IdoA2S-GlcNS6S repeating units have been defined as optimal ligands. Our data supports a model in which the RBD of the spike confers sequence specificity and an additional HS binding site in the S1/S2 proteolytic cleavage site 8 enhances the avidity of binding probably by non-specific interactions.
Although the IdoA2S-GlcNS6S is abundantly present in heparin, it is a minor component of HS. 20 Interestingly, it has been reported that the expression of the (GlcNS6S-IdoA2S)3 motif is highly regulated and plays a crucial role in cell behavior and disease including endothelial cell activation. 21 Severe thrombosis in COVID-19 patients is associated with endothelial dysfunction 22 and a connection may exist between SARS-CoV-2's ability to bind to HS and thrombotic disorder. Removal of the sulfate at C-3 of N-sulfoglucosamine (GlcNS3S) of the pentasaccharide results in a 10 5 -fold reduction in binding affinity. 25 Importantly, such a functionality is not present in the identified HS ligand of SARS-CoV-2 spike, and therefore compounds can be developed that can inhibit cell binding, but do not interact with ATIII. As a result, such preparations can be used at higher doses without causing adverse side effects. Our data also shows that multivalent interactions of the spike with HS results in high avidity of binding.
This observation provides opportunities to develop glycopolymers modified by HS oligosaccharides as to inhibitors of SARS-CoV-2 cell binding to prevent or treat COVID-