PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF) BINDS TO GLYCOSAMINOGLYCANS - ANALYSIS OF THE BINDING-SITE

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PE DF extractions from the interphotoreceptor matrix are more efficient w ith increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used af finity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sul fate columns and eluted from each with an increase in NaCl concentrati on. However, in the presence of urea, the protein lost its affinity fo r heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific bindi ng EC50 = 40 mu g/mL) and was sensitive to increasing NaCl concentrati ons. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysi ne residues by biotinylation lost its capacity for interacting with he parin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product co ntaining residues 21-similar to 260 bound to heparin with similar affi nity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3-and helix F that has a bas ic electrostatic surface potential and is densely populated with lysin es exposed to the surface (K134, K137, K189, K191, H212, and K214) tha t are available to interact with various glycosaminoglycans/polyanions . This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.