Abstract
Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.
Highlights
The physiological functions of Pigment epithelium-derived factor (PEDF) can be divided into neurotrophic/neuroprotective and antiangiogenic activities
The Susceptibility of PEDF to Limited Trypsin Proteolysis Was Increased by Heparin—To investigate putative structural changes induced by ligand binding, PEDF was titrated with heparin, hyaluronic acid, chondroitin sulfate, or collagen and studied by means of limited proteolysis
It has been suggested that the mechanism of enhanced receptor binding involves a structural change of PEDF [14]
Summary
Materials—Heparin, hyaluronic acid, chondroitin sulfate, heparinase, ovalbumin, porcine trypsin, heparin-BSA, and o-phenylenediamine dichloride were from Sigma. To analyze the effect of ionic strength, PEDF (2 g) was digested with trypsin at a ratio of 1:40 (w/w) in 25 l of 20 mM Tris-HCl, pH 7.4, containing increasing amounts of NaCl (0 –100 mM). The samples were digested with trypsin in a ratio of 1:1 (w/w) in the presence of increasing amounts of NaCl (100 –250 mM), in 20 l for 15 min at 23 °C before the addition of phenylmethylsulfonyl fluoride (2.5 mM final concentration). The effects of specific heparin size variants were analyzed by preincubating PEDF (2 g) and tetrasaccharides (dp4), octasaccharides (dp8), or hexadecasaccharides (dp16) at 37 °C in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl. After 1 h the samples were digested with trypsin (enzyme:substrate ratio 1:1, w/w) for 15 min in 20 l at 23 °C. Wells were developed as described above and all data points were collected in duplicates
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