Shedding of Membrane-associated Mucin MUC16 at Ocular Su

眼苏膜相关粘蛋白 MUC16 的脱落

• 批准号: 6992596
• 负责人: TIMOTHY D BLALOCK
• 金额: $ 4.83万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-29 至 2008-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6992596
• 关键词: apical membrane; cell biology; cell line; endopeptidases; enzyme activity; epithelium; eye; gene expression; human genetic material tag; membrane activity; mucins; postdoctoral investigator; protein engineering; protein structure; protein structure function; recombinant proteins; tear; tissue /cell culture; transfection

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.

描述（由申请人提供）：本申请的目的是获得有关膜相关黏液蛋白MUC16在眼表上的生理作用及其脱落对正常功能的影响的信息。初步研究表明MUC16在眼表上皮顶端细胞层（微褶）的膜褶皱尖端表达。此外，MUC16的可溶性形式和几种蛋白酶已在人泪液样品中检测到。由于在靠近细胞膜的MUC16胞外结构域上存在一个假定的蛋白酶裂解位点，因此在眼表上皮上可能存在MUC16在眼表脱落的机制。拟议的研究将利用截断的双标记MUC16构建体在培养细胞中表达。一旦该蛋白在细胞表面表达，将开发体外试验，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外畴脱落。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，例如剩余胞内结构域的命运，细菌对细胞表面的粘附以及表面结构的变化。我们假设MUC16的外畴从眼表上皮脱落，并且在人泪膜中发现的蛋白酶反应下其脱落增加，从而导致正常生理功能的改变。