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.
