O-Glycans on Mucins at the Ocular Surface

描述(申请人提供)：粘蛋白是一个大的和严重的O-糖基化的糖蛋白家族，由所有湿表面上皮合成，包括角膜和结膜上皮。眼表上皮细胞产生分泌的粘蛋白MUC5AC和膜相关粘蛋白MUC的1、4和16。粘蛋白上的末端碳水化合物是接触细胞外环境最多的，但人们对它们在眼表每种粘蛋白中的作用和性质知之甚少。我们之前的研究已经证明，干眼症患者MUC16上针对末端碳水化合物的特异性抗体的结合发生了变化。这项建议的目的是确定个别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-聚糖在维持眼表湿润和防止病原体入侵方面的作用。目的I：我们假设个别粘蛋白被眼表上皮细胞不同地O-糖基化。我们建议：A.描述从正常人泪液中分离出的单个粘蛋白上的末端O-连接碳水化合物的特征。确定并定位负责其生物合成的糖基转移酶(唾液酸基转移酶)。C.体外测试糖萼粘蛋白上的末端O-连接碳水化合物在赋予上皮细胞去粘附性和对腺相关病毒的转染抵抗力方面的作用。目的II：我们假设在干眼症患者中，膜相关粘蛋白的末端O-糖链结构发生改变，从而影响眼表的水化。我们建议：a.确定干眼症患者泪膜表面纯化的每一种膜相关粘蛋白的末端碳水化合物是否发生改变。确定干眼症患者糖基转移酶(唾液酸基转移酶)的表达是否发生改变。在体外确定角膜和结膜细胞中粘蛋白的末端碳水化合物的消耗是否增强了孟加拉玫瑰对这些细胞的穿透性。目的III：我们假设泪液中分泌的粘蛋白MUC5AC上的O-连接碳水化合物与铜绿假单胞菌具有特异性的亲和力，防止它们附着在眼表糖萼上，促进它们的清除。我们建议：a.将铜绿假单胞菌的结合与从眼泪中收集的单个粘蛋白的O-连接碳水化合物进行比较。在体外确定膜相关粘蛋白上O-糖链合成的抑制如何影响铜绿假单胞菌对角膜细胞的黏附。

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.