Chang Vincent, Mahoney Keira E, Lian Isaac, Chen Ryan, Chung Nara, Utheim Tor Paaske, Karlsson Niclas G, Malaker Stacy A
Department of Chemistry, Yale University, New Haven CT, USA.
Department of Medical biochemistry, Oslo University Hospital, Oslo, Norway.
J Biol Chem. 2025 Aug 8:110580. doi: 10.1016/j.jbc.2025.110580.
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.
泪液由多种细胞外糖蛋白组成，这些糖蛋白对眼内稳态至关重要。在泪液糖蛋白组中，乳铁蛋白高度表达，并在免疫反应、泪液分泌和抗菌活性中发挥关键作用。重要的是，糖基化占乳铁蛋白分子量的50%以上。然而，尽管如此，关于乳铁蛋白上的特定聚糖结构以及它们如何影响其蛋白质折叠、功能或下游生物学过程，我们一无所知。同样，乳铁蛋白聚糖的改变是否与眼部疾病相关仍然完全未知。为了填补这一知识空白，我们利用质谱（MS）对泪液进行了首次O-糖蛋白组学研究。在此，我们报告了乳铁蛋白糖基化前所未有的覆盖范围，详细描述了19个带有多种聚糖结构的O-糖基化位点。此外，我们利用Alphafold 3.0和GlycoShape来可视化这些聚糖对其结构的影响，证明O-糖基化使蛋白质主链刚性且伸展。令人惊讶的是，我们还检测到了两种乳铁蛋白剪接异构体的蛋白质水平证据，这是通过质谱首次观察到这些异构体。同时，我们描述了迄今为止泪液糖蛋白组最全面的特征，阐明了免疫球蛋白A（IgA）、乳铁蛋白和其他具有临床相关性作为诊断生物标志物的糖蛋白的糖基化谱。总体而言，这项研究为未来泪液糖蛋白的生化研究及其作为眼部疾病诊断或治疗工具的应用奠定了关键基础。
