As CLQ and CLQ-OH are potential therapies for SARS-CoV-2 infection, it is important to check whether the amino acid residues identified as critical for ganglioside binding are conserved among clinical isolates. The alignment of the 111–162 domain of 11 clinical isolates of SARS-CoV-2 from various geographic origins (including Asia and USA) is shown in Fig. 10 . In this region, which contains the ganglioside-binding domain identified in the present report, all amino acids are fully conserved. Interestingly, the motif is built like a giant consensus ganglioside-binding domain: a central region displaying the critical aromatic residue (Phe-135) and a basic residue at each end (Lys-113 and Arg-158). In the middle of each stretch separating this typical triad, there is a N-glycosylation site (Asn-122 and Asn-149). These last regions are not directly involved in ganglioside binding, so the oligosaccharide linked to these asparagine residues could be perfectly intercalated between the sugar head group of gangliosides.
Fig. 10 Amino acid sequence alignments of the ganglioside-binding domain (GBD) of the SARS-CoV-2 spike protein. (a) Clinical SARS-CoV-2 isolates aligned with the reference sequence (6VSB_A, fragment 111–162). The amino acid residues involved in GM1 binding are indicated in red. Two asparagine residues acting as glycosylation sites are highlighted in yellow. (b) Alignments of human and animal viruses compared with SARS-CoV-2 (6VSB_A, fragment 111–162). Deletions are highlighted in green, amino acid changes in residues involved in ganglioside binding are highlighted in blue, conserved residues of the GBD are highlighted in red, and asparagine residues acting as glycosylation sites are highlighted in yellow.