O-glycosylation affects proteolysis of the native tear peptidome

immune response and participating in antimicrobial activity. The biological function of these native peptides is governed by their biochemical properties including peptide length, charge, and hydrophobicity. Accordingly, the production of native peptides within the tear film is tightly regulated through a complex interplay between circulating proteases and the proteolytic susceptibility of the protein substrate. Though glycosylation has been shown to regulate the proteolysis of specific proteins in vitro, the extent to which endogenous cleavage motifs are mediated by proximal O-glycans remains unexplored in a more complex sample. Furthermore, the existence and identity of native tear peptides bearing O-glycans has yet to be elucidated, largely due to the high analytical complexity of tear fluid and the difficulty in characterizing O-glycosylated peptides. To address this gap, we leveraged advances in mass spectrometry (MS) to provide the first observation of circulating tear O-glycopeptides, detailing their biochemical properties and overall glycan compositions. Beyond systematic profiling of the O-glycopeptidome, we employed site-specific glycoproteomic analysis followed by molecular dynamics to investigate the interplay between O-glycan proximity and proteolysis. Here, we observed that O-glycans preferentially occupy glycosites distal from the N-/C-terminus, thereby influencing distinct cleavage motifs and peptide backbones. Finally, we showed in silico that O-glycosylation can mediate the solvent accessibility of proximal cleavage residues, thus providing a structural basis for these observations. Taken together, this study defines the proteolytic landscape at the ocular surface and highlights a potential role for tear fluid O-glycans.