Shedding of the Membrane-associated Mucin MUC16 at the Ocular Surface

描述（由申请方提供）：本提案的目的是获得关于眼表膜相关粘蛋白MUC16的生理作用以及脱落对正常功能的影响的信息。初步研究表明，MUC16表达于眼表上皮细胞顶端细胞层（微褶）的膜褶皱顶端。此外，在人泪液样品中检测到MUC16和几种蛋白酶的可溶形式。由于推定的蛋白酶切割位点存在于细胞膜附近的MUC16细胞外结构域上，因此很可能在眼表面上皮上存在MUC16在眼表面上脱落的机制。拟议的研究将利用截短的双标记MUC16构建体在培养细胞中表达。一旦这种蛋白质在细胞表面上表达，将开发体外测定以使用切割其他膜相关粘蛋白的蛋白酶和泪膜中发现的蛋白酶来定量MUC16胞外域脱落。最后，将测量MUC16胞外域从细胞表面脱落对几个过程的影响，例如剩余胞内结构域的命运、细菌粘附到细胞表面以及表面结构的变化。我们假设MUC16的胞外域从眼表上皮脱落，并且其脱落响应于人泪膜中发现的蛋白酶而增加，这导致正常生理功能的改变。

DESCRIPTION (provided by applicant): The objective of this proposal is to obtain information regarding the physiological role of the membrane associated mucin MUC16 on the ocular surface and the effects that shedding have on normal function. Preliminary studies have shown that MUC16 is expressed at the tips of membrane folds on the apical cell layer (microplicae) on the ocular surface epithelium. Also, soluble forms of MUC16 and several proteases have been detected in samples of human tear fluid. Since a putative protease cleavage site exists on the MUC16 extracellular domain near the cell membrane, it is likely that a mechanism of MUC16 shedding on the ocular surface exists on the ocular surface epithelium. The proposed studies will utilize a truncated, dual-labeled MUC16 construct for expression in cultured cells. Once this protein is expressed on the cell surface, in vitro assays will be developed to quantitate MUC16 ectodomain shedding using both proteases that cleave other membrane-associated mucins and proteases found in the tear film. Finally, the effects of MUC16 ectodomain shedding from the cell surface on several processes will be measured, such as the fate of the remaining intracellular domain, bacterial adherence to the cell surface, and changes in surface architecture. We hypothesize that the ectodomain of MUC16 is shed from the ocular surface epithelium and its shedding increases in response to proteases found in human tear film, which leads to an alteration in normal physiological function.