O‑Glycosylation Shapes the Proteolytic Landscape of the Native Tear Peptidome.

Authors

Vincent Chang, Ryan J Chen, Isaac Lian, Keira E Mahoney, Stacy A Malaker

Published in

JACS Au. Volume 6. Issue 7. Pages 4321-4335. Jul 27, 2026. Epub Jun 22, 2026.

Abstract

The endogenous tear peptidome plays essential roles in maintaining ocular homeostasis by stimulating 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 complex biofluid. Furthermore, the existence and identity of native tear peptides bearing O-glycans have 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 extracellular 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 correlation between O-glycan proximity and proteolysis. Here, we observed that O-glycans preferentially occupy glycosites distal from the N-/C-terminus, which was associated with distinct cleavage motifs and peptide backbones. We then showed in silico that O-glycosylation on lacritin can mediate the solvent accessibility of proximal cleavage residues, thus providing a structural basis for these observations. Finally, cleavage studies of glycosylated and nonglycosylated lacritin revealed that the unmodified form exhibited significantly greater degradation by endogenous tear proteases, supporting a protective role for its glycans. Taken together, this study defines the proteolytic landscape at the ocular surface and highlights a potential role for tear fluid O-glycans in mediating proteolysis.

PMID:
42529429
Bibliographic data and abstract were imported from PubMed on 17 Sep 2026.

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