O-Glycans on Mucins at the Ocular Surface

描述（由申请人提供）：粘膜表面，包括眼表面，被上皮覆盖，这对防止损伤和感染至关重要。细胞表面相关黏蛋白是该上皮顶端糖萼的主要成分，延伸至质膜以上500 nm处，远远超过所有其他膜相关蛋白和脂质。它们表现出大量的o -聚糖链，这些o -聚糖链与蛋白质骨干中的氨基酸串联重复序列相连，从而在局部产生高浓度的碳水化合物配体。细胞表面糖基化的改变与眼表面上皮疾病（如干眼症）中粘膜屏障的破坏有关，但导致这种破坏的机制大多尚未确定。在上一个资助期内，我们确定了半乳糖凝集素-3是人类眼表上皮表达的主要碳水化合物结合蛋白。半乳糖凝集素-3是一种多聚体凝集素，可以交联和寡聚蛋白显示多个低聚糖链。这一发现使我们推测，多价O-聚糖结合半乳糖凝集素-3寡聚物，在角膜和结膜细胞糖萼起伏的膜脊上方形成强大且高度组织的凝集素-碳水化合物晶格。在本应用中，我们拟确定糖萼中的粘蛋白型o -聚糖是否与半乳糖凝集素-3结合，在眼表面形成一个顶细胞表面屏障，以及这个屏障是否阻止外来物质通过质膜流入，阻止微生物和炎症介质进入顶细胞表面受体。最后，我们建议确定在眼表上皮疾病患者中是否存在o -聚糖：凝集素屏障的分子破坏。了解包括细胞表面糖萼成分在内的上皮防御机制有助于开发旨在恢复眼表疾病患者上皮屏障成分的新治疗方法。

DESCRIPTION (provided by applicant): Mucosal surfaces, including those of the ocular surface, are covered by epithelium that is critical to preventing damage and infection. Cell surface-associated mucins are major components on the apical glycocalyx of this epithelium, extending up to 500 nm above the plasma membrane, far above all other membrane-associated proteins and lipids. They exhibit large numbers of O-glycan chains tethered to tandem repeats of amino acids in their protein backbone that create a locally high concentration of carbohydrate ligands. Alteration of cell surface glycosylation has been associated with the disruption of the mucosal barrier in ocular surface epithelial disease (e.g., dry eye), but the mechanisms that lead to this disruption remain mostly uncharacterized. During the last funding period, we identified galectin-3 as the major carbohydrate-binding protein expressed by human ocular surface epithelia. Galectin-3 is a multimeric lectin that can cross-link and oligomerize proteins displaying multiple oligosaccharide chains. This finding led us to hypothesize that multivalent O- glycans bind galectin-3 oligomers to form strong and highly organized lectin-carbohydrate lattices above the undulating membrane ridges in corneal and conjunctival cells' glycocalyx. In this application, we propose to determine whether mucin-type O-glycans in the glycocalyx associate with galectin-3 to form an apical cell surface barrier on the ocular surface, and whether this barrier prevents the influx of foreign substances through the plasma membrane and the access of microorganisms and inflammatory mediators to apical cell surface receptors. Finally, we propose to determine whether in patients with ocular surface epithelial disease there is a molecular breach of the O-glycan:galectin barrier. Understanding the mechanism of epithelial defense that includes components of the cell surface glycocalyx could help with the development of new therapeutic approaches aimed to restore components of the epithelial barrier in patients with ocular surface disease. PUBLIC HEALTH RELEVANCE: Mucosal surfaces such as the corneal and conjunctival epithelia of the eye are faced with the critical function of forming a protective barrier that prevents cellular damage and systemic infection, while allowing the exchange of molecules with the extracellular milieu. In this application, we propose to determine the role of mucin carbohydrates (O-glycans) and carbohydrate-binding proteins (galectins) in the formation of this barrier on apical cells of the ocular surface epithelia. The understanding of the O-glycan:galectin barrier, which has not been previously described, may help in the clinical treatment of diseases in which the ocular surface is compromised.