O-Glycans on Mucins at the Ocular Surface

描述（由申请方提供）：粘蛋白是由所有湿表面上皮细胞（包括角膜和结膜上皮细胞）合成的大的和高度O-糖基化的糖蛋白家族。眼表面上皮细胞产生分泌的粘蛋白MUC 5AC和膜相关粘蛋白MUC 1、4和16。粘蛋白的末端碳水化合物是最暴露于细胞外环境，但很少有人知道他们的角色和字符在每个粘蛋白在眼表。我们以前的研究已经证明，在干眼症患者中，MUC 16上的末端碳水化合物特异性抗体的结合改变。该提案旨在鉴定个体眼粘蛋白上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。目的一：我们假设，个别粘蛋白的差异O-糖基化的眼表上皮细胞。我们建议：A.表征从正常个体泪液中分离的单个粘蛋白上的末端O-连接碳水化合物的库。B。鉴定和定位负责其生物合成的糖基转移酶（唾液酸转移酶）。C.体外测试糖萼粘蛋白上末端O-连接的碳水化合物在赋予上皮细胞解粘特性和抵抗腺相关病毒转染中的作用。AIM II：我们假设在干眼患者中，膜相关粘蛋白的末端O-聚糖结构发生改变，从而影响眼表的水合作用。我们建议：A.确定从干眼症患者的顶端泪液表面纯化的每种膜相关粘蛋白的末端碳水化合物是否存在改变。B。确定干眼患者中糖基转移酶（唾液酸转移酶）的表达是否有改变。C.在体外确定角膜和结膜细胞中粘蛋白末端碳水化合物的消耗是否会增强虎红进入这些细胞的渗透率。AIM III：我们假设泪液中分泌的粘蛋白MUC 5AC上存在的O-连接的碳水化合物对铜绿假单胞菌具有特异性亲和力，从而防止它们附着于眼表面糖萼并促进它们的清除。我们建议：A.比较铜绿假单胞菌与从泪液中收集的单个粘蛋白的O-连接碳水化合物的结合。B。在体外确定O-聚糖合成对膜相关粘蛋白的抑制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.