Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.