N-glycosylation and ocular surface homeostasis

项目摘要/摘要 眼表上皮细胞屏障功能的破坏与一系列疾病有关， 包括干眼-一种影响全球数百万人的与年龄相关的疾病，其药理作用 治疗方法仍未解决。角膜结膜终末分化的复层上皮细胞 通过由跨膜粘蛋白组成的特殊保护结构维持屏障功能 一组高度糖基化的蛋白质，具有极大的胞外结构域。半乳糖 粘蛋白多糖上的残基通过多聚体蛋白Galectin-3交联在顶端糖萼上 防止细胞损伤。防止细胞功能和动态平衡下降的关键是 氨基己糖途径--一系列合成N-糖链增加的合成反应 先驱物。氨基己糖途径的激活导致内侧N-葡聚糖分支增加 高尔基体和可调节细胞分化的细胞表面Galectin-3晶格的形成。重要的是 补充氨基己糖途径的代谢物也可以提高蛋白质质量。 控制机制。尽管有这些关键作用，N-糖基化和蛋白水解酶功能的相关性 羟色胺代谢产物在眼表健康和疾病中的作用仍未得到充分研究。我们假设N- 葡聚糖在眼表上皮细胞中具有动态作用，在炎症和驱动下发生变化 粘蛋白屏障和应激反应。这项建议的长期目标是确定贡献 N-葡聚糖支化在促进眼表健康中的作用，以及氨基己糖是否被激活 途径可用于眼睛的治疗收益。以下具体目标将针对这一目标： (1)研究人角膜上皮细胞黏蛋白N-糖链及其与Galectin-3的关系。 正常和促炎状态，(2)确定N-葡聚糖促进屏障的调节作用 功能和减少眼表面的应激反应，以及(3)评估是否激活 氨基己糖途径促进眼表动态平衡。这项研究将解决在很大程度上未被研究的 N-葡聚糖在眼表屏障功能和应激反应中的作用，并探索其潜在的 药物激活氨基己糖途径治疗眼表疾病。

Project Summary/Abstract Disruption of barrier function at the ocular surface epithelia is associated with a wide range of disorders that includes dry eye—an age-related disease affecting millions of people worldwide and whose pharmacological treatment remains unresolved. Terminally differentiated stratified epithelial cells in cornea and conjunctiva maintain barrier function through a specialized protective structure composed of transmembrane mucins, a group of heavily glycosylated proteins characterized by extremely large extracellular domains. Galactosyl residues on mucin glycans are cross-linked on the apical glycocalyx by the multimeric protein galectin-3 to prevent cellular damage. Critical to preventing the decline in cellular function and homeostasis is the hexosamine pathway—a series of anabolic reactions that generate increased synthesis of N-glycan precursors. Activation of the hexosamine pathway leads to increased branching of N-glycans in the medial Golgi and the formation of cell-surface galectin-3 lattices that can modulate cell differentiation. Importantly, metabolic supplementation with hexosamine pathway metabolites is also known to enhance protein quality control mechanisms. Despite these critical roles, the relevance of N-glycosylation and the function of hesoxamine metabolites in ocular surface health and disease remains understudied. We hypothesize that N- glycans have a dynamic role in ocular surface epithelial cells, changing in response to inflammation and driving mucin barrier and stress responses. The long-term objective of this proposal is to determine the contribution of the N-glycan branching in promoting ocular surface health, and whether activation of the hexosamine pathway can be used for therapeutic gain in the eye. The following specific aims will address this objective: (1) to characterize mucin N-glycans in human corneal epithelial cells and their relationship to galectin-3 under normal and pro-inflammatory conditions, (2) to determine the regulatory role of N-glycans in promoting barrier function and reducing stress responses at the ocular surface, and (3) to evaluate whether activation of the hexosamine pathway promotes ocular surface homeostasis. This research will address the largely unstudied function of N-glycans in ocular surface barrier function and stress responses, and explore the potential of pharmacologically activating the hexosamine pathway for the treatment of ocular surface diseases.