Maspin in the Cornea

描述(申请人提供)：bMaspin是丝氨酸蛋白酶抑制物超家族的成员，主要研究其在癌细胞中的肿瘤抑制功能。像许多上皮细胞一样，正常的角膜上皮细胞和内皮细胞合成maspin。令人惊讶的是，正常的人角膜基质细胞也会合成maspin，这是已知的唯一能合成这种分子的非上皮细胞类型。在培养中，随着传代的角膜基质细胞发展出类似伤口成纤维细胞的特性，它们失去了合成maspin的能力，这让人想起转移性癌细胞的损失。然而，角膜基质成纤维细胞对外源性maspin的反应是通过增加与I型和IV型胶原、纤维连接蛋白和层粘连蛋白的黏附而实现的。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、防止肿瘤发生和血管生成等事件中发挥关键作用。这一建议特别针对这一假设，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性增加需要maspin的多个结构域，并由maspin与细胞表面分子结合启动。本项目的主要目标是确定maspin分子生物学活性所需的区域，并确定角膜基质成纤维细胞上maspin结合伙伴(S)的特征。这项建议的目的如下： 1)确定maspin的哪些区域是诱导培养的人基质成纤维细胞与ECM分子黏附增加所必需的。将使用rMaspin-卵清蛋白杂合蛋白、maspin定点突变体、多肽和/或针对特定maspin表位的抗体来探索maspin的功能结构域。 2)鉴定maspin与人角膜基质成纤维细胞和细胞外基质分子的结合，并确定maspin的哪个区域(S)需要结合。Maspin与角膜基质细胞和选定的ECM分子的相互作用将被表征，maspin结构域的确定将集中在失去功能的maspin-卵清蛋白杂交体上，结果将通过maspin结合位点多肽得到证实。 3)鉴定和鉴定角膜基质成纤维细胞膜上的maspin结合分子S。Maspin结合蛋白(S)将通过maspin交联分离，其蛋白水解肽将通过MALDI-TOF分析和/或N-端序列分析进行鉴定。它们将被克隆，在酵母系统中表达，并通过下拉试验和交联性研究表征它们与maspin的相互作用。

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.