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

描述(申请人提供)：这项建议的目的是获得有关膜相关粘蛋白MUC16在眼表面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(微皱褶)的膜折尖端。此外，在人类泪液样本中也检测到了可溶性的MUC16和几种蛋白酶。由于在靠近细胞膜的MUC16胞外区存在一个假定的蛋白酶裂解位点，因此MUC16在眼表上的脱落机制可能存在于眼表上皮细胞上。拟议的研究将利用截短的、双标记的MUC16结构在培养细胞中表达。一旦这种蛋白在细胞表面表达，将开发体外检测方法来定量MUC16胞外结构域的脱落，使用裂解其他膜相关粘蛋白的蛋白酶和在泪膜中发现的蛋白酶。最后，将测量MUC16胞外结构域从细胞表面脱落对几个过程的影响，例如剩余细胞内结构域的命运、细菌在细胞表面的黏附以及表面结构的变化。我们假设MUC16的胞外结构域是从眼表上皮细胞中脱落的，并且它的脱落随着人泪膜中发现的蛋白水解酶的增加而增加，从而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.