Maspin in the Cornea

角膜内的 Maspin

• 批准号: 6508796
• 负责人: Sally S. Twining
• 金额: $ 28.78万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6508796
• 关键词: SDS polyacrylamide gel electrophoresis; binding sites; cell adhesion; chimeric proteins; collagen; corneal stroma; crosslink; enzyme inhibitors; extracellular matrix; fibroblasts; fibronectins; human tissue; laminin; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; organ culture; polymerase chain reaction; protein binding; protein biosynthesis; protein protein interaction; protein sequence; protein structure function; serine proteinases; site directed mutagenesis

DESCRIPTION (provided by applicant): bMaspin is a member of the serine proteinase inhibitor superfamily that has been studied primarily for its tumor suppressor function in carcinoma cells. Like many epithelial cells, normal corneal epithelial and endothelial cells synthesize maspin. Surprisingly, normal human corneal stromal cells also synthesize maspin, the only non-epithelial cell type known to synthesize this molecule. In culture, as passaged corneal stromal cells develop properties resembling wound fibroblasts, they lose their ability to synthesize maspin, reminiscent of the loss by metastatic carcinoma cells. However, corneal stromal fibroblasts respond to treatment with exogenous maspin by increased adhesion to types I and IV collagens, fibronectin and laminin. Studying maspin's function in the cornea is particularly important since its ability to regulate matrix attachment and cell motility may play a critical role in such events as regulation of stromal wound healing and prevention of tumorigenesis and angiogenesis. This proposal specifically addresses the hypothesis that the increase in adhesion of corneal stromal fibroblasts to type I and type IV collagens, laminin and fibronectin requires multiple domains of maspin and is initiated by maspin binding to a cell surface molecule. The major goal of this project is to identify the regions of the maspin molecule required for its biological activity and to characterize maspin binding partner(s) on corneal stromal fibroblasts. The aims of this proposal are the following: 1) To determine which regions of maspin are required for the induction of increased adhesion of cultured human stromal fibroblasts to ECM molecules. The functional maspin domains will be explored using rMaspin-ovalbumin hybrid proteins, site-specific maspin mutants, peptides and/or antibodies to specific maspin epitopes. 2) To characterize maspin binding to human corneal stromal fibroblasts and selected ECM molecules and determine which region(s) of maspin is required for binding. Maspin interaction with corneal stromal cells and selected ECM molecules will be characterized and the maspin domains determined by focusing on the loss-of-function maspin-ovalbumin hybrids and the results confirmed using maspin binding site peptides. 3) To identify and characterize the maspin-binding molecule(s) on corneal stromal fibroblast membranes. Maspin binding proteins(s) will be isolated by maspin cross-linking and their proteolytic peptides identified by MALDI-TOF analysis and/or N-terminal sequence analysis. They will be cloned, expressed in a yeast system and their interactions with maspin characterized using pull down assays and cross-linking studies.

描述（由申请人提供）：bMaspin是丝氨酸蛋白酶抑制剂超家族的成员，主要研究其在癌细胞中的肿瘤抑制功能。和许多上皮细胞一样，正常角膜上皮细胞和内皮细胞也能合成巨噬蛋白。令人惊讶的是，正常的人类角膜基质细胞也能合成maspin，这是已知唯一能合成这种分子的非上皮细胞类型。在培养中，当传代的角膜基质细胞发展出类似创面成纤维细胞的特性时，它们失去了合成巨噬蛋白的能力，这让人想起转移癌细胞的丧失。然而，角膜基质成纤维细胞对外源性巨噬蛋白的反应是通过增加对I型和IV型胶原、纤维连接蛋白和层粘连蛋白的粘附。研究maspin在角膜中的功能尤为重要，因为它调节基质附着和细胞运动的能力可能在调节基质伤口愈合和预防肿瘤发生和血管生成等事件中发挥关键作用。这一建议特别提出了一个假设，即角膜基质成纤维细胞对I型和IV型胶原、层粘连蛋白和纤维连接蛋白的粘附增加需要masmasin的多个结构域，并且是由masmasin与细胞表面分子结合引发的。该项目的主要目标是确定maspin分子的生物活性所需的区域，并表征maspin在角膜间质成纤维细胞上的结合伙伴。这项建议的目的如下：