O-Glycans on Mucins at the Ocular Surface

描述（由申请方提供）：粘液表面，包括眼表的粘液表面，被上皮覆盖，这对防止损伤和感染至关重要。细胞表面相关粘蛋白是该上皮的顶端糖萼上的主要组分，延伸至质膜上方500 nm，远高于所有其他膜相关蛋白和脂质。它们在其蛋白质骨架中表现出大量的O-聚糖链与氨基酸的串联重复序列相连，从而产生局部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（例如，干眼症），但导致这种破坏的机制仍然大多未被表征。在上一个资助期间，我们确定了半乳糖凝集素-3作为主要的碳水化合物结合蛋白表达的人眼表上皮细胞。半乳糖凝集素-3是一种多聚体凝集素，可以交联和寡聚化显示多个寡糖链的蛋白质。这一发现使我们假设多价0-聚糖结合半乳糖凝集素-3低聚物以在角膜和结膜细胞的糖萼中的波状膜脊上方形成坚固且高度组织化的凝集素-碳水化合物晶格。在本申请中，我们建议确定糖萼中的粘蛋白型O-聚糖是否与半乳糖凝集素-3结合以在眼表面上形成顶端细胞表面屏障，以及该屏障是否阻止外来物质通过质膜流入以及微生物和炎症介质进入顶端细胞表面受体。最后，我们建议，以确定是否在眼表上皮疾病的患者有一个分子突破的O-聚糖：半乳糖凝集素屏障。了解上皮防御机制，包括细胞表面糖萼的成分，可以帮助开发新的治疗方法，旨在恢复眼表疾病患者的上皮屏障成分。 公共卫生相关性：诸如眼睛的角膜和结膜上皮的粘膜表面面临形成保护屏障的关键功能，该保护屏障防止细胞损伤和全身性感染，同时允许分子与细胞外环境交换。在本申请中，我们建议确定粘蛋白碳水化合物（O-聚糖）和碳水化合物结合蛋白（半乳糖凝集素）在眼表上皮细胞顶端细胞形成这种屏障中的作用。对O-聚糖：半乳糖凝集素屏障的理解，这是以前没有描述过的，可能有助于临床治疗眼表受损的疾病。

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.