Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry

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 might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind 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 GAGbinding 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. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2

“…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.…”

“…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.…”

Contribution of syndecans to the cellular entry of SARS-CoV-2

“…According to current reports, this heparin-binding motif may facilitate SARS-CoV-2 host cell entry 36 .…”

“…However, scienti c evidence shows that endocytosis of SARS-CoV also occurs through a novel, clathrinand caveolae-independent endocytic pathway, mediated by attachment to cell surface heparan sulfate proteoglycans (HSPGs) [29][30][31][32] . Meanwhile, it has been also revealed that the S1 subunit of the SARS-CoV-2 spike protein, the subunit responsible for cellular attachment, contains the heparin-binding core motif PRRAR [33][34][35][36] ( Supplementary Fig. S1).…”

Increased Expression of Chondroitin Sulfotransferases following AngII may Contribute to Pathophysiology Underlying Covid-19 Respiratory Failure: Impact may be Exacerbated by Decline in Arylsulfatase B Activity

“…Studies of coronavirus-receptor interactions have implicated the sulfated glycosaminoglycans heparin and heparan sulfate in viral attachment to mammalian cells [1,2]. Recent work on the structure of the spike protein of SARS-CoV-2 has provided detail about the glycosylations of the spike protein and interactions between the spike protein and carbohydrates [3,4]. Findings suggest potential for both proteincarbohydrate and carbohydrate-carbohydrate interactions between the spike glycoprotein and cell receptors.…”