O-Glycans on Mucins at the Ocular Surface

描述(申请人提供)：粘膜表面，包括那些眼睛表面，被上皮覆盖，这是防止损伤和感染的关键。细胞表面相关粘蛋白是该上皮顶端糖萼上的主要成分，在质膜上方延伸至500 nm，远远超过所有其他膜相关蛋白和脂类。它们表现出大量的O-糖链，与蛋白质骨架中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。在眼表上皮病(如干眼)中，细胞表面糖基化的改变与粘膜屏障的破坏有关，但导致这种破坏的机制大多尚不清楚。在上一次资助期间，我们确定Galectin-3是人眼表面上皮细胞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，可以使多个寡糖链上的蛋白质发生交联和寡聚。这一发现使我们假设，多价O-多糖与Galectin-3寡聚体结合，在角膜和结膜细胞的糖基化上形成强大且高度有序的凝集素-碳水化合物晶格。在这一应用中，我们建议确定糖萼中的粘液型O-糖链是否与Galectin-3结合形成眼表面的顶端细胞表面屏障，以及该屏障是否阻止外来物质通过质膜进入以及微生物和炎症介质对顶端细胞表面受体的访问。最后，我们建议确定在眼表上皮病患者中是否存在O-糖蛋白：半乳糖屏障的分子断裂。了解上皮防御的机制，包括细胞表面糖基化的成分，有助于开发新的治疗方法，旨在恢复眼表疾病患者的上皮屏障成分。 与公共卫生相关：眼部的粘膜表面，如角膜和结膜上皮，面临着形成一种保护屏障的关键功能，该屏障可以防止细胞损伤和全身感染，同时允许与细胞外环境进行分子交换。在这一应用中，我们建议确定粘蛋白碳水化合物(O-多糖)和碳水化合物结合蛋白(Galectins)在眼表上皮细胞顶端细胞形成这种屏障中的作用。对O-葡聚糖：半乳糖屏障的了解，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.