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Galectin-3 binds isoform- and glycoform-specific lacritin in tear fluid

ABSTRACT: Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, cell migration, autophagy, and signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins across the epithelial cell glycocalyx, forming lattice structures important for barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoproteins in tears to affect downstream biological processes has yet to be investigated. As increased Gal-3 levels in tears are known to correlate with ocular pathologies such as dry eye disease (DED), we sought to elucidate the Gal-3 interactome in tear fluid and uncover biological insights into the function of Gal-3 beyond adhesion to the epithelial cell surface. Here, we combined ELISA, lectin affinity enrichment, mass spectrometry (MS) glycoproteomics, and lectin blotting to uncover Gal-3 interactors and their associated glycoepitopes. Overall, we report nearly 100 proteins enriched from tear fluid across 3 different patients, identifying proteins involved in immune response, inflammation, and antimicrobial activity. Most notably, we report lacritin as a novel ligand for Gal-3 and demonstrate that specific glycoforms of lacritin bearing core 2 O-glycans preferentially engage with Gal-3. Lastly, we show that the Gal-3-lacritin axis is spliceoform-specific and dependent on lacritin multimerization. Taken together, this study elucidates new ligands for Gal-3 in tear film and reveals mRNA splicing and multimerization as new biochemical mechanisms which fine-tune Gal-3 binding events.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tear

SUBMITTER: Vincent Chang

LAB HEAD: Stacy Malaker

PROVIDER: PXD071066 | Pride | 2026-04-22

REPOSITORIES: pride

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Source:

			Action	DRS
	250729_VC_ctrl_tears_1.raw	Raw
	250729_VC_ctrl_tears_2.raw	Raw
	250729_VC_ctrl_tears_3.raw	Raw
	250808_VC_G3_1_DTT_IAA_postelution.raw	Raw
	250808_VC_G3_2_DTT_IAA_postelution.raw	Raw

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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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Galectin-3 binds isoform- and glycoform-specific lacritin in tear fluid

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