Maspin in the Cornea

角膜内的 Maspin

• 批准号: 6631459
• 负责人: Sally S. Twining
• 金额: $ 30万
• 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/6631459
• 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，该成员主要是为其在癌细胞中肿瘤抑制功能而进行的。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。出乎意料的是，正常的人角膜基质细胞还合成Maspin，Maspin是唯一已知合成该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类似于伤口成纤维细胞的特性，它们失去了合成maspin的能力，使人联想到转移性癌细胞的损失。然而，角膜基质成纤维细胞通过增加对I型和IV胶原，纤连蛋白和层粘连蛋白的粘附而对外源Maspin的治疗作出反应。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起关键作用。该建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需要多个MASPIN的粘附粘附，并由Maspin结合与细胞表面分子结合。该项目的主要目的是确定Maspin分子其生物活性所需的区域，并在角膜基质成纤维细胞上表征Maspin结合伴侣。该提案的目的是以下： 1）确定诱导培养的人基质成纤维细胞对ECM分子的粘附增加需要哪些MASPIN区域。功能性maspin结构域将使用RMASPIN-OVERBUMIN杂化蛋白，位点特异性Maspin突变体，肽和/或特定Maspin表位抗体进行探索。 2）表征与人角膜基质成纤维细胞和选定的ECM分子结合的MASPIN结合，并确定结合需要哪个MASPIN区域。 Maspin与角膜基质细胞和选定的ECM分子的相互作用将被表征，并通过关注功能丧失的Maspin-overbumin杂种来确定的Maspin结构域，并使用Maspin结合位点肽证实了结果。 3）识别和表征角膜基质成纤维细胞膜上的maspin结合分子。 Maspin结合蛋白（S）将通过Maspin交联及其通过MALDI-TOF分析和/或N末端序列分析鉴定的蛋白水解肽分离。它们将被克隆，在酵母系统中表达，并且它们与MASPIN的相互作用，这些相互作用使用下拉测定和交联研究来表征。