A glycosaminoglycan microarray identifies the binding of SARS‐CoV‐2 spike protein to chondroitin sulfate E

Abstract

Heparan sulfate (HS), a sulfated glycosaminoglycan (GAG), was reported to be a necessary host attachment factor that promotes SARS‐CoV‐2 infection. In this study, we developed GAG microarrays based on fluorescence detection for high‐sensitivity screening of the GAG‐binding specificity of proteins and applied it for the analysis of SARS‐CoV‐2 spike (S) protein. Among the 20 distinct GAGs, the S protein bound not only to heparin (HEP)/HS but also to chondroitin sulfate E (CSE) in a concentration‐dependent manner. We then analyzed the specificity of each subunit of the S protein. While the S1 subunit showed exclusive binding to HEP, the S2 subunit also bound to CSE and HEP/HS. CSE might act as an alternative attachment factor for HS in SARS‐CoV‐2 infection.

The diverse role of heparan sulfate and other GAGs in SARS-CoV-2 infections and therapeutics

“…On the other hand, ACE2 receptor-binding to S-protein is not inhibited by sulfated galactofucan and glucuronomannan ( Jin et al, 2020 ), and kappa-carrageenan and lambda-carrageenan were less effective than iota-carrageenan ( Morokutti-Kurz et al, 2021 ). A low binding affinity of the virus was observed towards CS ( Dwivedi et al, 2021 ; Song et al, 2020 ; Tandon et al, 2021 ; Watanabe et al, 2021 ), DS ( Dwivedi et al, 2021 ), or HA ( Liu et al, 2021 ). CS, however, might act as alternative binding partner for the S2 subunit throughout infection ( Watanabe et al, 2021 ).…”

“…This points towards the presence of several GAG-binding sites on the S-protein beyond just the RBD. A study by Watanabe et al (2021) revealed specific binding patterns of full monomeric S-protein and the S1 and S2 subunits. While S1 almost exclusively bound to heparin, S2 showed high affinities for CS and HS and their fractions.…”

“…Two types of key experiments were conducted to evaluate GAG-mediated inhibition of SARS-CoV-2 infection. In one experimental approach protein-GAG-interactions are studied by using surface plasmon resonance spectrometry (SPR) or structurally defined oligosaccharide sequences in a microarray high throughput format ( Hao et al, 2021 ; Liu et al, 2021 ; Watanabe et al, 2021 ; Yu et al, 2020 ). Both strategies take advantage of competitive binding of SARS-CoV-2 or the S-protein to heparins in the presence of other GAGs, which allows for rapid screening studies.…”

“…Commonly applied methods for structural determination are nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and X-ray crystallography ( Yang & Chi, 2017 ). Oligosaccharides with structurally defined sequences are often used in a microarray high throughput format to study specific interactions with proteins ( Hao et al, 2021 ; Liu et al, 2021 ; Watanabe, Takeda, Hiemori, Minamisawa, & Tateno, 2021 ; Yu et al, 2020 ). Other popular methods, used in affinity studies of protein-GAG-interactions, are surface plasmon resonance spectrometry (SPR), affinity chromatography or isothermal calorimetry ( Shi et al, 2021 ).…”

Two years into COVID‐19 – Lessons in SARS‐CoV‐2 and a perspective from papers in FEBS Letters

“…Akin to many other viruses, SARS‐CoV‐2 binds to multiple cell surface molecules, including receptors and attachment factors, such as heparan sulphate (HS), a sulphated glycosaminoglycan (GAG). Watanabe and colleagues developed highly sensitive fluorescence‐based GAG microarrays and identified the binding of SARS‐CoV‐2 S‐protein to chondroitin sulphate E (CSE) and confirmed the binding to HS [22]. Intriguingly, S1 bound to heparin and S2 to both CSE and HS (as well as heparin).…”