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O-glycosylation affects proteolysis of the native tear peptidome

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 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

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tear

SUBMITTER: Vincent Chang

LAB HEAD: Stacy Malaker

PROVIDER: PXD072578 | Pride | 2026-07-08

REPOSITORIES: Pride

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Source:

			Action	DRS
	250903_VC_tear_peptide_1.raw	Raw
	250903_VC_tear_peptide_2.raw	Raw
	250909_VC_tear_peptide_pooled_1.raw	Raw
	Lacritin_PRP_endogenous_glycopeps_250903_VC_tear_peptide_1.raw.xlsx	Xlsx
	Lacritin_PRP_endogenous_glycopeps_250903_VC_tear_peptide_2.raw.xlsx	Xlsx

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Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.

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O-glycosylation affects proteolysis of the native tear peptidome

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