In-depth analysis of the tear fluid glycoproteome reveals diverse lacritin glycosylation and spliceoforms

Abstract Tear fluid comprises a diverse group of extracellular glycoproteins which are critical for ocular homeostasis. Within the tear fluid glycoproteome, lacritin is highly expressed and plays a key role in immune response, tear secretion, and antimicrobial activity. Importantly, glycosylation constitutes over 50% of lactritin’s molecular weight. However, despite this fact, nothing is known about the specific glycan structures on lacritin and how they influence its protein folding, function, or downstream biological processes. Similarly, it remains completely unknown whether alterations to lacritin glycans are correlated with ocular pathologies. To address this gap in knowledge, we harnessed mass spectrometry (MS) to conduct the first O-glycoproteomic study of tear fluid. Here, we report unprecedented coverage of lacritin glycosylation, detailing 19 O-glycosites bearing a myriad of glycan structures. Further, we leveraged Alphafold 3.0 and GlycoShape to visualize the impact of these glycans on its structure, demonstrating that O-glycosylation renders the protein backbone rigid and extended. Surprisingly, we also detected protein-level evidence of two lacritin spliceoforms, representing the first observation of these isoforms by MS. Simultaneously, we describe the most comprehensive characterization of the tear fluid glycoproteome to date, elucidating the glycosylation profile of Immunoglobulin A (IgA), lactoferrin, and other glycoproteins with demonstrated clinical relevance as diagnostic biomarkers. Overall, this study lays critical groundwork for future biochemical investigation of tear fluid glycoproteins and their application as diagnostic or therapeutic tools for ocular diseases. Competing Interest Statement S.A.M. is a co-inventor on a Stanford patent related to the use of mucinases as research tools. Abbreviations - ACN - Acetonitrile - ETD - Electron transfer dissociation - EThcD - Electron transfer/higher-energy collision dissociation - HCD - Higher-energy collisional dissociation - DED - Dry eye disease - TGM2 - Transglutaminase 2 - IgA1 - ImmunoglobulinA1 - IgJ - Immunoglobulin J chain - PBS - Phosphate Buffered Saline - T antigen - Galβ1-3GalNAcα1-Ser/Thr - Tn antigen - GalNAcα1-Ser/Thr. Type 3 H-antigen (Fuc α1-2-Galβ1-3GalNAcα1-Ser/Thr)