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S.S. Twining, C. Ngamkitidechakul, W.J. O'Brien, J.M. Burke; Sufficiency of the Reactive Site Loop of Maspin for Induction of Keratocyte-Matrix Adhesion: Conversion of Ovalbumin to a Maspin-Like Molecule . Invest. Ophthalmol. Vis. Sci. 2003;44(13):871.
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Purpose:Maspin is an ov-serpin that regulates adhesion, cell migration and invasion of epithelial cells. Not only do corneal epithelial and endothelial cells synthesize maspin but corneal stromal cells synthesize this molecule. Upon passage or immortalization of human corneal stromal cells, synthesis of maspin is down regulated but maspin still induces adhesion of these cells to collagens type I and IV, laminin and fibronectin. The purpose of this study was to determine the portion of the maspin molecule required for the stimulation of adhesion of keratocytes to type I collagen. Methods:The reactive site loop region (RSL), the C-terminal or both the RSL and C-terminal regions of maspin were replaced by the corresponding region of ovalbumin or the same regions of maspin were swapped into ovalbumin using overlap PCR. Maspin and these swap mutants were expressed as N-terminal His-tagged proteins in yeast using the alpha factor leader sequence secretion system and purified on a nickel column. Maspin, the mutants, ovalbumin or a peptide containing the 15 RSL residues were incubated with immortalized or passaged human corneal stromal cells for 18 hrs. The cells were released with EDTA and allowed to adhere to collagen type I coated wells. Following washing, the adherent cells were stained with crystal violet and adhesion quantified. Results:Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen. Maspin with ovalbumin as the C-terminal region retained activity. Substitution of the maspin RSL into ovalbumin converted the inactive ovalbumin into a fully active molecule. The RSL peptide alone was also capable of enhancing cell-matrix adhesion. Conclusions: These studies demonstrate the induction of increased adhesion of cultured corneal stomal cells requires only the 15 amino acid RSL for activity.
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