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. 2003 Aug 15;374(Pt 1):199–206. doi: 10.1042/BJ20030313

Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization.

Steen V Petersen 1, Zuzana Valnickova 1, Jan J Enghild 1
PMCID: PMC1223568  PMID: 12737624

Abstract

Pigment epithelium-derived factor (PEDF) inhibits the formation of blood vessels in the eye by inducing apotosis in actively dividing endothelial cells. The activity of PEDF equals or supersedes that of other anti-angiogenic factors, including angiostatin, endostatin and thrombospondin-1. In addition, PEDF has the potential to promote the survival of neurons and affect their differentiation. Here we show that PEDF is present in plasma at a concentration of approx. 100 nM (5 microg/ml) or twice the level required to inhibit aberrant blood-vessel growth in the eye. Thus the systemic delivery of PEDF has the potential to affect angiogenesis or neurotrophic processes throughout the body, significantly expanding the putative physiological role of the protein. A complete map of all post-translational modifications revealed that authentic plasma PEDF carries an N-terminal pyroglutamate blocking group and an N-linked glycan at position Asn266. The pyroglutamate residue may regulate the activity of PEDF analogously to the manner in which it regulates thyrotropin-releasing hormone.

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Selected References

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  1. Abraham G. N., Podell D. N. Pyroglutamic acid. Non-metabolic formation, function in proteins and peptides, and characteristics of the enzymes effecting its removal. Mol Cell Biochem. 1981 Aug 11;38(Spec No)(Pt 1):181–190. doi: 10.1007/BF00235695. [DOI] [PubMed] [Google Scholar]
  2. Araki T., Taniwaki T., Becerra S. P., Chader G. J., Schwartz J. P. Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death. J Neurosci Res. 1998 Jul 1;53(1):7–15. doi: 10.1002/(SICI)1097-4547(19980701)53:1<7::AID-JNR2>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  3. Becerra S. P., Palmer I., Kumar A., Steele F., Shiloach J., Notario V., Chader G. J. Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. J Biol Chem. 1993 Nov 5;268(31):23148–23156. [PubMed] [Google Scholar]
  4. Berg T., Leth-Larsen R., Holmskov U., Højrup P. Structural characterisation of human proteinosis surfactant protein A. Biochim Biophys Acta. 2000 Nov 30;1543(1):159–173. doi: 10.1016/s0167-4838(00)00184-9. [DOI] [PubMed] [Google Scholar]
  5. Birn H., Verroust P. J., Nexo E., Hager H., Jacobsen C., Christensen E. I., Moestrup S. K. Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein. J Biol Chem. 1997 Oct 17;272(42):26497–26504. doi: 10.1074/jbc.272.42.26497. [DOI] [PubMed] [Google Scholar]
  6. Chader G. J. PEDF: Raising both hopes and questions in controlling angiogenesis. Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2122–2124. doi: 10.1073/pnas.061024098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crawford S. E., Stellmach V., Ranalli M., Huang X., Huang L., Volpert O., De Vries G. H., Abramson L. P., Bouck N. Pigment epithelium-derived factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell antitumor activity. J Cell Sci. 2001 Dec;114(Pt 24):4421–4428. doi: 10.1242/jcs.114.24.4421. [DOI] [PubMed] [Google Scholar]
  8. Cummins P. M., O'Connor B. Pyroglutamyl peptidase: an overview of the three known enzymatic forms. Biochim Biophys Acta. 1998 Dec 8;1429(1):1–17. doi: 10.1016/s0167-4838(98)00248-9. [DOI] [PubMed] [Google Scholar]
  9. Dawson D. W., Volpert O. V., Gillis P., Crawford S. E., Xu H., Benedict W., Bouck N. P. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science. 1999 Jul 9;285(5425):245–248. doi: 10.1126/science.285.5425.245. [DOI] [PubMed] [Google Scholar]
  10. DiPaolo B. R., Pignolo R. J., Cristofalo V. J. Identification of proteins differentially expressed in quiescent and proliferatively senescent fibroblast cultures. Exp Cell Res. 1995 Sep;220(1):178–185. doi: 10.1006/excr.1995.1304. [DOI] [PubMed] [Google Scholar]
  11. Duh Elia J., Yang Hoseong S., Suzuma Izumi, Miyagi Masaru, Youngman Elaine, Mori Keisuke, Katai Miyuki, Yan Lin, Suzuma Kiyoshi, West Karen. Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth. Invest Ophthalmol Vis Sci. 2002 Mar;43(3):821–829. [PubMed] [Google Scholar]
  12. Hu G. F. Fluorophore-assisted carbohydrate electrophoresis technology and applications. J Chromatogr A. 1995 Jun 23;705(1):89–103. doi: 10.1016/0021-9673(95)93203-8. [DOI] [PubMed] [Google Scholar]
  13. Hutchings Helen, Maitre-Boube Martine, Tombran-Tink Joyce, Plouët Jean. Pigment epithelium-derived factor exerts opposite effects on endothelial cells of different phenotypes. Biochem Biophys Res Commun. 2002 Jun 21;294(4):764–769. doi: 10.1016/S0006-291X(02)00548-X. [DOI] [PubMed] [Google Scholar]
  14. Jackson P. Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device. Anal Biochem. 1991 Aug 1;196(2):238–244. doi: 10.1016/0003-2697(91)90460-b. [DOI] [PubMed] [Google Scholar]
  15. Kozaki K., Miyaishi O., Koiwai O., Yasui Y., Kashiwai A., Nishikawa Y., Shimizu S., Saga S. Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells. J Biol Chem. 1998 Jun 12;273(24):15125–15130. doi: 10.1074/jbc.273.24.15125. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  18. Meyer Christina, Notari Luigi, Becerra S. Patricia. Mapping the type I collagen-binding site on pigment epithelium-derived factor. Implications for its antiangiogenic activity. J Biol Chem. 2002 Sep 16;277(47):45400–45407. doi: 10.1074/jbc.M208339200. [DOI] [PubMed] [Google Scholar]
  19. Miller E. J., Rhodes R. K. Preparation and characterization of the different types of collagen. Methods Enzymol. 1982;82(Pt A):33–64. doi: 10.1016/0076-6879(82)82059-4. [DOI] [PubMed] [Google Scholar]
  20. Mosimann S. C., Ardelt W., James M. N. Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity. J Mol Biol. 1994 Mar 4;236(4):1141–1153. doi: 10.1016/0022-2836(94)90017-5. [DOI] [PubMed] [Google Scholar]
  21. Ozols J. Amino acid analysis. Methods Enzymol. 1990;182:587–601. doi: 10.1016/0076-6879(90)82046-5. [DOI] [PubMed] [Google Scholar]
  22. Pignolo R. J., Cristofalo V. J., Rotenberg M. O. Senescent WI-38 cells fail to express EPC-1, a gene induced in young cells upon entry into the G0 state. J Biol Chem. 1993 Apr 25;268(12):8949–8957. [PubMed] [Google Scholar]
  23. Seprodi J., Coy D. H., Vilchez-Martinez J. A., Pedroza E., Huang W. Y., Schally A. V. Cyclic analogues of luteinizing hormone-releasing hormone with significant biological activities. J Med Chem. 1978 Sep;21(9):993–995. doi: 10.1021/jm00207a029. [DOI] [PubMed] [Google Scholar]
  24. Shevchenko A., Wilm M., Vorm O., Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1;68(5):850–858. doi: 10.1021/ac950914h. [DOI] [PubMed] [Google Scholar]
  25. Simonovic M., Gettins P. G., Volz K. Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor. Proc Natl Acad Sci U S A. 2001 Sep 18;98(20):11131–11135. doi: 10.1073/pnas.211268598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Singer A. J., Clark R. A. Cutaneous wound healing. N Engl J Med. 1999 Sep 2;341(10):738–746. doi: 10.1056/NEJM199909023411006. [DOI] [PubMed] [Google Scholar]
  27. Singh V. K., Chader G. J., Rodriguez I. R. Structural and comparative analysis of the mouse gene for pigment epithelium-derived factor (PEDF). Mol Vis. 1998 Apr 20;4:7–7. [PubMed] [Google Scholar]
  28. Steele F. R., Chader G. J., Johnson L. V., Tombran-Tink J. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1526–1530. doi: 10.1073/pnas.90.4.1526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stellmach V., Crawford S. E., Zhou W., Bouck N. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci U S A. 2001 Jan 23;98(5):2593–2597. doi: 10.1073/pnas.031252398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stratikos E., Alberdi E., Gettins P. G., Becerra S. P. Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells. Protein Sci. 1996 Dec;5(12):2575–2582. doi: 10.1002/pro.5560051220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tombran-Tink J., Chader G. G., Johnson L. V. PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity. Exp Eye Res. 1991 Sep;53(3):411–414. doi: 10.1016/0014-4835(91)90248-d. [DOI] [PubMed] [Google Scholar]
  32. Volpert Olga V., Zaichuk Tetiana, Zhou Wei, Reiher Frank, Ferguson Thomas A., Stuart P. Michael, Amin Mohammad, Bouck Noel P. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat Med. 2002 Apr;8(4):349–357. doi: 10.1038/nm0402-349. [DOI] [PubMed] [Google Scholar]
  33. Wu Y. Q., Becerra S. P. Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing. Invest Ophthalmol Vis Sci. 1996 Sep;37(10):1984–1993. [PubMed] [Google Scholar]
  34. Wu Y. Q., Notario V., Chader G. J., Becerra S. P. Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes. Protein Expr Purif. 1995 Aug;6(4):447–456. doi: 10.1006/prep.1995.1060. [DOI] [PubMed] [Google Scholar]

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