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S Becerra, RN Fariss, C Meyer; Characterization of the Pigment Epithelium-derived Factor and Collagen Interactions . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2407.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: PEDF is an extracellular serpin that inhibits angiogenesis in the cornea and vitreous of the mammalian eye. Collagen is a major extracellular component of the cornea and vitreous. The purpose of this study is to probe for direct binding of PEDF to collagens and analyzed its binding site. Methods: Binding to collagen was performed by size-exclusion ultrafiltration, radioligand-binding assays, ligand blotting and ligand-affinity column chromatography. Collagen was detected by slot and western blotting with antibodies to collagen I, II, III; and PEDF by immunohistochemistry and western blotting. Recombinant human PEDF (rhuPEDF) was chemically modified with fluorescein-5-EX, succinimidyl ester. Soluble corneal and vitreal extracts were prepared by differential centrifugation from bovine eyes. Results: PEDF was detected in the bovine cornea and vitreous. rhuPEDF interacted with corneal and vitreal collagens. It formed specific complexes with collagen I, II and III, with type I having the highest affinity. 125I-labeled PEDF bound to immobilized collagen I in a specific and saturable fashion (kd = 133nM). Unlabeled PEDF and a PEDF fragment (amino acid positions 44-418) competed efficiently with 125I-PEDF for binding to collagen I. Angiostatin, endostatin and a synthetic PEDF peptide (positions 78-121; 44-mer) did not compete. The binding to collagen was sensitive to increasing NaCl concentrations, implying an ionic nature for the PEDF-collagen interactions. Given that the surface of the PEDF protein has two distinct areas of opposite ionic potentials, we used Fl-PEDF, chemically modified on positively charged lysine residues, to identify the PEDF ionic region that interacts with collagen. Fl-PEDF bound to collagen I and required ≷5-times higher concentrations of NaCl to prevent binding, compared to unmodified protein. This implied an increase in the collagen binding affinity when lysines were blocked. Conclusion: PEDF showed binding affinity for collagens mediated by ionic interactions. A region with a negative ionic potential on the surface of the PEDF protein, rich in aspartic and glutamic acids, constitutes a proposed collagen-binding site for PEDF and the first described for a serpin. The collagen-binding property of PEDF may be very important in surface localization of its antiangiogenic effects to the cornea and vitreous, and play a role in inhibition of adhesion, migration and/or cell proliferation during neovascularization.
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