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

Area of Science:

• Ophthalmology and Vision Science
• Immunology and Glycobiology
• Biochemistry and Molecular Biology

Background:

• Galectin-3 (Gal-3) is a key protein involved in inflammation, immune responses, and cell signaling.
• At the ocular surface, Gal-3 crosslinks mucins, essential for barrier function.
• The role of Gal-3 in tear fluid and its interactions with tear glycoproteins are largely unknown, despite its association with dry eye disease (DED).

Purpose of the Study:

• To investigate the Gal-3 interactome in human tear fluid.
• To identify novel biological functions of Gal-3 in the tear film beyond epithelial cell adhesion.
• To explore the molecular mechanisms, including glycoepitopes and protein modifications, governing Gal-3 interactions in tears.

Main Methods:

• Enzyme-linked immunosorbent assay (ELISA) and lectin blotting were employed.
• Lectin affinity enrichment coupled with mass spectrometry (MS)-based glycoproteomics identified Gal-3 binding partners.
• Analysis focused on tear fluid from multiple patients to ensure robust findings.

Main Results:

• Nearly 100 proteins interacting with Gal-3 were identified in tear fluid.
• These proteins are significantly involved in immune response, inflammation, and antimicrobial activities.
• Lacritin was identified as a novel Gal-3 ligand, with binding preference for specific glycoforms (core 2 O-glycans) and dependence on spliceoforms and multimerization.

Conclusions:

• This study elucidates novel Gal-3 ligands within the tear film, expanding our understanding of its function in ocular surface health.
• The findings reveal that mRNA splicing and protein multimerization are critical regulatory mechanisms for Gal-3 binding.
• The identified Gal-3-lacritin axis offers new insights into the pathogenesis of ocular surface diseases like DED.