SARS-CoV-2 viruses
engage ACE2 as a functional receptor
with their
spike protein. The S1 domain of the spike protein contains a C-terminal
receptor binding domain (RBD) and an N-terminal domain (NTD). The
NTD of other coronaviruses includes a glycan binding cleft. However,
for the SARS-CoV-2 NTD, protein–glycan binding was only observed
weakly for sialic acids with highly sensitive methods. Amino acid
changes in the NTD of variants of concern (VoC) show antigenic pressure,
which can be an indication of NTD-mediated receptor binding. Trimeric
NTD proteins of SARS-CoV-2, alpha, beta, delta, and omicron did not
reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2
beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was
sensitive to sialidase pretreatment. Glycan microarray analyses identified
a putative 9-O-acetylated sialic acid as a ligand,
which was confirmed by catch-and-release ESI-MS, STD-NMR analyses,
and a graphene-based electrochemical sensor. The beta (501Y.V2-1)
variant attained an enhanced glycan binding modality in the NTD with
specificity toward 9-O-acetylated structures, suggesting
a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was
quickly selected against. These results indicate that SARS-CoV-2 can
probe additional evolutionary space, allowing binding to glycan receptors
on the surface of target cells.