O-Glycans on Mucins at the Ocular Surface

描述（由申请人提供）：粘蛋白是由所有湿表面上皮（包括角膜和结膜上皮）合成的大且高度o糖基化的糖蛋白家族。眼表上皮产生分泌性黏液蛋白MUC5AC和膜相关黏液蛋白MUC's 1、4和16。黏液蛋白上的末端碳水化合物是最易暴露于细胞外环境的，但人们对它们在眼表黏液中的作用和特征知之甚少。我们之前的研究表明，在干眼症患者中，MUC16末端碳水化合物特异性抗体的结合发生了改变。本研究旨在确定单个眼粘蛋白的末端碳水化合物结构、参与其生物合成的酶以及末端o -聚糖在维持湿润眼表和防止病原体入侵中的作用。目的1：我们假设单个粘蛋白在眼表上皮中存在o糖基化差异。我们建议：A.表征从正常人泪液中分离的单个粘蛋白上末端o链碳水化合物的全部结构。鉴定和定位负责其生物合成的糖基转移酶（唾液基转移酶）。C.在体外测试糖萼粘蛋白上末端o连接碳水化合物在赋予上皮细胞分离特性和抵抗腺相关病毒转染方面的作用。AIM II：我们假设在干眼症患者中，膜相关黏蛋白的末端o -聚糖结构发生改变，影响眼表面的水合作用。我们建议：A.确定从干眼症患者的根尖撕裂表面纯化的每种膜相关粘蛋白的末端碳水化合物是否有改变。B.确定干眼症患者中糖基转移酶（唾液转移酶）的表达是否有改变。C.体外确定角膜和结膜细胞黏蛋白末端碳水化合物的消耗是否会增强玫瑰红蛋白进入这些细胞的外显率。AIM III：我们假设泪液中分泌的粘蛋白MUC5AC上存在的o键碳水化合物对铜绿假单胞菌具有特异性亲和力，阻止它们附着在眼表糖萼上，并促进它们的清除。我们建议：A.比较P. aeruginosa与从眼泪中收集的单个粘蛋白的o键碳水化合物的结合。B.体外确定抑制膜相关粘蛋白上的o聚糖合成如何影响铜绿假单胞菌对角膜细胞的粘附。

DESCRIPTION (provided by applicant): Mucins are a family of large and heavily O-glycosylated glycoproteins synthesized by all wet-surfaced epithelia, including the corneal and conjunctival epithelia. The ocular surface epithelia produce the secreted mucin MUC5AC and the membrane-associated mucins MUC's 1, 4 and 16. Terminal carbohydrates on mucins are the most exposed to the extracellular milieu, but little is known about their roles and character in each mucin at the ocular surface. Our previous studies have demonstrated that binding of an antibody specific to a terminal carbohydrate on MUC16 is altered in dry eye patients. This proposal is aimed at identifying terminal carbohydrate structures on individual ocular mucins, the enzymes involved in their biosynthesis and the role of terminal O-glycans in maintaining a wet ocular surface and preventing pathogen invasion. AIM I: We hypothesize that individual mucins are differentially O-glycosylated by the ocular surface epithelia. We propose to: A. Characterize the repertoire of terminal O-linked carbohydrates on individual mucins isolated from tears of normal individuals. B. Identify and localize glycosyltransferases (sialyltransferases) responsible for their biosynthesis. C. Test in vitro the role of terminal O-linked carbohydrates on mucins of the glycocalyx in conferring disadhesive properties to epithelial cells and resistance to transfection with adeno-associated viruses. AIM II: We hypothesize that in dry eye patients there is an alteration in terminal O-glycan structures of the membrane-associated mucins that affects hydration of the ocular surface. We propose to: A. Determine if there is an alteration of terminal carbohydrates of each membrane-associated mucin purified from the apical tear surface of dry eye patients. B. Determine if there is an alteration in the expression of glycosyltransferases (sialyltransferases) in dry eye patients. C. Determine in vitro whether the depletion of terminal carbohydrates on mucins in corneal and conjunctival cells enhances the penetrance of rose bengal into these cells. AIM III: We hypothesize that O-linked carbohydrates present on the secreted mucin MUC5AC in the tear fluid have specific affinity to P. aeruginosa, preventing their attachment to the ocular surface glycocalyx and facilitating their clearance. We propose to: A. Compare the binding of P. aeruginosa to the O-linked carbohydrates of individual mucins collected from tears. B. Determine in vitro how inhibition of O-glycan synthesis on membrane-associated mucins affects P. aeruginosa adherence to corneal cells.