Structural Basis of a High-Affinity Antibody Binding to Glycoprotein with Glycocalyx Decay.

Cancer-associated defects in mucin-type O-glycosylation expose truncated glycans on cell-surface glycoproteins and create tumor-associated glycopeptide epitopes. However, the structural principles by which antibodies recognize specific MUC1 glycopeptide sequences remain incompletely defined. Here, we report the molecular basis of MUC1 recognition by 16A, a high-affinity antibody isolated against hypoglycosylated MUC1. Co-crystal structures of 16A Fab bound to site-selectively glycosylated MUC1 peptides revealed that 16A recognizes an STAPPAHG-centered epitope within the MUC1 variable number tandem repeat region. Surface plasmon resonance analysis showed that GalNAc modification at the threonine site in this sequence increased Fab binding affinity by 30.6-fold relative to the nonglycosylated peptide. Structural and mutational analyses identified a hydrogen bond between the threonine-linked GalNAc and Trp34 in heavy-chain CDR1 as a key contributor to this affinity enhancement. Cell-based binding assays further showed that COSMC deficiency enhanced 16A recognition of MUC1-expressing cells, while molecular dynamics simulations suggested that Ser-linked Core 1 extension can create local steric incompatibility near CDRH1 Arg32. Together, these results define a peptide-register-driven recognition mode in which the MUC1 peptide backbone determines specificity, whereas a site-compatible GalNAc acts as an affinity-enhancing element at the edge of the paratope. This structural framework may guide the design of MUC1 glycopeptide-based vaccines and antibody therapeutics.