Site‐specific O‐glycans influence lacritin structure and multimerization in tears

Abstract Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C‐terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O‐glycans that comprise more than 50% of its molecular weight remains largely unknown. To address this gap, we leveraged mass spectrometry (MS)‐based glycoproteomics, AlphaFold 3.0, and molecular dynamics (MD) to explore the structural role of site‐specific O‐glycans on C‐terminal lacritin. In doing so, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Based on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O‐glycans may participate in intramolecular glycan–protein interactions that influence its structure and the spatial arrangement of Lys101 and Lys104. Differences in the solvent accessible surface area (SASA) and root mean squared fluctuation (RMSF) of these residues further suggested that proximal O‐glycosylation could affect their ability to participate in crosslinking. To test these predictions, we performed in vitro crosslinking assays and demonstrated reduced TGM2‐mediated multimerization of glycosylated versus unmodified lacritin. Finally, we show that recombinant glycoforms bearing O‐glycan patterns similar to endogenous lacritin multimers are enriched after TGM2 crosslinking. Taken together, these findings underscore a central role for lacritin O‐glycans in affecting structural topology and multimerization with implications for its downstream biological activity.