Mapping galectin-3 ligands in human tear fluid establishes spliceoform-dependent lacritin binding.

Galectin-3 (Gal-3) is a carbohydrate-binding protein which plays crucial roles in inflammation, immune response, and cell signaling. At the ocular surface, Gal-3 is also known to crosslink transmembrane mucins to support barrier function. However, the biological role of Gal-3 in circulating tear fluid remains largely unexplored. Similarly, whether Gal-3 engages extracellular glycoprotein ligands in tears to affect biological processes is unknown. Herein, we combine lectin ELISA, affinity enrichment, mass spectrometry (MS)-based 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 ligand for Gal-3 and demonstrate that their binding is spliceoform-specific and dependent on lacritin multimerization. Taken together, this study elucidates new ligands for Gal-3 in tear film and investigates new biochemical mechanisms that fine-tune Gal-3 binding events.