ABSTRACT
Consecutive glycosylation sites occur in both self and viral proteins. Glycan-shielding of underneath peptide region is a double-edged sword, that avoids immune attack to self-proteins, but helps viruses including HIV-1 and SARS-CoV2 to escape antibody binding. Here we report a high-affinity antibody, 16A, binding to linear peptide containing consecutive glycosylation sites. Co-crystallization of 16A Fab and glycopeptides with GalNAc modifications at different sites showed that STAPPAHG is the sequence recognized by 16A antibody. GalNAc modification at Threonine site on STAPPAHG sequence significantly increased the affinity of Fab binding by 30.6 fold (KD=6.7nM). The increased affinity is conferred by hydrophilic and pi-stacking interactions between the GalNAc residue on Threonine site and a Trp residue from the CDR1 region of the heavy chain. Furthermore, molecular modeling suggested that GalNAc on T site causes more favorable conformation for antibody binding. These results showed that glycan modification most proximal to linear peptide core epitope significantly increases antigenicity of a glycopeptide epitope. The antibody recognition mode by peptide-binding CDR groove with a glycan-binding edge, may shed light on designing of linear glycopeptide-based vaccines for cancer and viral diseases. Teaser A high-affinity antibody was found to bind densely glycosylated glycoprotein region by a peptide binding groove of the antibody’s variant region, with a glycan-binding edge specific to glycosylation site most proximal to core peptide epitope.