Maspin in the Cornea

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。与许多上皮细胞一样，正常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合成maspin，这是已知合成这种分子的唯一非上皮细胞类型。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丧失。然而，角膜基质成纤维细胞对外源性maspin治疗的反应是与I型和IV型胶原、纤连蛋白和层粘连蛋白的粘附增加。研究maspin在角膜中的功能特别重要，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起关键作用。该提议特别解决了以下假设：角膜基质成纤维细胞与I型和IV型胶原、层粘连蛋白和纤连蛋白的粘附增加需要maspin的多个结构域，并且由maspin与细胞表面分子结合启动。该项目的主要目标是确定maspin分子的生物活性所需的区域，并表征角膜基质成纤维细胞上的maspin结合伴侣。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分子粘附增加所必需的。将使用rMaspin-卵清蛋白杂合蛋白、位点特异性Maspin突变体、针对特异性Maspin表位的肽和/或抗体来探索功能性Maspin结构域。 2)表征maspin与人角膜基质成纤维细胞和选定ECM分子的结合，并确定结合所需的maspin区域。将表征Maspin与角膜基质细胞和所选ECM分子的相互作用，并通过关注功能丧失的maspin-卵清蛋白杂合体来确定maspin结构域，并使用maspin结合位点肽来确认结果。 3)鉴定和表征角膜基质成纤维细胞膜上的maspin结合分子。通过maspin交联分离Maspin结合蛋白，并通过MALDI-TOF分析和/或N-末端序列分析鉴定其蛋白水解肽。它们将被克隆，在酵母系统中表达，并使用下拉测定和交联研究表征它们与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.