Computational analysis reveals non-consensus N-glycosylation sequons in antibody Fab region

Protein glycosylation at asparagine typically occurs at a consensus motif. However, recent studies have reported instances of N-glycosylation at non-consensus sites, though the mechanisms and implications of these atypical modifications remain unclear. In this study, we identified novel non-consensus N-glycosylation motifs with low glycosylation occupancy in the Fab region of human antibodies. We developed a computational workflow to predict the interaction between non-consensus peptides and the eukaryotic oligosaccharyltransferase (OST) complex. This model was validated through site-directed mutagenesis around the asparagine residue and glycosylation quantification via mass spectrometry. Our results show that glycan occupancy at non-consensus sites can be modulated by mutations that influence OST binding affinity. Pharmacological inhibition of OST activity reduced non-consensus and consensus glycosylation in both Fab and Fc regions. Additionally, we identified new non-consensus glycosylation sites in natural human antibodies, revealing the sequence preferences governing these modifications. These findings provide mechanistic insights into OST sequence specificity and establish a computational and analytical framework for assessing atypical N-glycosylation, aiding glycan profile control in therapeutic antibody development.

Antibody structure; fab glycosylation; glycan analysis; intravenous immunoglobulin; mass spectrometry; non-consensus N-glycosylation; oligosaccharyltransferase; peptide docking.