Heparan Sulfate Microarray Lends Insight into Selective Binding of SARS‐CoV‐2 Spike Glycoprotein

Chittum, John; Sankaranarayanan, Nehru; O'Hara, Connor; Desai, Umesh

SARS‐CoV‐2 remains a prominent threat to human lives despite the recent approval of vaccines and antibodies across the world. There is also a growing concern of mutations in the virus that demand effective strategies to combat infection. SARS‐CoV‐2 infects cells via its surface Spike glycoprotein (SgP), a homo‐trimeric assembly of S1 and S2 subunits, that binds to cell surface angiotensin‐converting enzyme 2 (ACE2), thereby gaining viral entry. SgP also utilizes the cell surface bound heparan sulfate proteoglycans (HSPGs) to enhance the efficiency of viral entry. The receptor‐binding domain (RBD) in the S1 subunit interacts with both the ACE2 receptor as well as the heparan sulfate (HS) chains of HSPGs. HS is a type of glycosaminoglycan (GAG) that has been recognized as a crucial factor in the infectivity of numerous viruses. Our early work suggested that of the numerous structural possibilities, 3‐O‐sulfated (3‐OS) sequences of HS may be involved in better recognition of SgP (bioRxiv (2020) DOI:10.1101/2020.10.08.331751). Large‐scale computational analysis based on our in‐house developed genetic algorithm‐based dual filtering strategy indicated that HS more favorably interacts with the RBD compared to other electropositive regions in the SgP trimer. The results suggested that the RBD of SgP prefers to recognize optimal three‐dimensional factors governed by chain length and sulfation pattern of HS sequences. Microarray screening of 24 distinct HS sequences against the S1 and RBD domains resulted in only eight sequences displaying reasonable affinity for the RBD, which were significantly weaker for the S1 subunit. Of these, two containing 3‐O­S sequences exhibited some of the highest signals on the array. Competition studies using the same microarray in the presence of fondaparinux (a 3‐OS‐containing pentasaccharide) and HS06 (a non‐3‐OS variant of a HS hexasaccharide) led to the observation that HS06 is a more efficient competitor than fondaparinux. Advanced computational experiments indicated that RBD tends to bind more effectively with 3‐OS‐containing HS chains when more than one 3‐OS groups is present. In conclusion, our work provides additional insight into the structural requirements within HS for mediating efficient viral entry, while also offering new avenues for developing potential inhibitors of SARS‐CoV‐2 infection.