Crystal Structure of Human PEDF, a Potent Anti- Angiogenic and Neurite Growth-Promoting Factor
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Crystal structure of human PEDF, a potent anti- angiogenic and neurite growth-promoting factor Miljan Simonovic*, Peter G. W. Gettins*, and Karl Volz†‡ Departments of *Biochemistry and Molecular Biology, and †Microbiology and Immunology, College of Medicine, University of Illinois, Chicago, IL 60612-7334 Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 26, 2001 (received for review May 30, 2001) Pigment epithelium-derived factor (PEDF), a noninhibitory member cornea leads to loss of visual acuity, opacification, and abnormal of the serpin superfamily, is the most potent inhibitor of angio- healing (24, 25). The neurotrophic and anti-angiogenic proper- genesis in the mammalian ocular compartment. It also has neuro- ties of PEDF argue in favor of its possible therapeutic value for trophic activity, both in the retina and in the central nervous pathologic ocular neovascularization and retinoblastoma treat- system, and is highly up-regulated in young versus senescent ment and perhaps more widely for control of neovascularization fibroblasts. To provide a structural basis for understanding its associated with tumor growth. many biological roles, we have solved the crystal structure of Although initial studies on the mechanism(s) of action of glycosylated human PEDF to 2.85 Å. The structure revealed the PEDF as a neurotrophic agent have been carried out based on organization of possible receptor and heparin-binding sites, and a hypothetical model of the molecule (26, 27), a more detailed showed that, unlike any other previously characterized serpin, approach has been greatly hindered by the absence of a three- PEDF has a striking asymmetric charge distribution that might be dimensional structure. We have now determined the crystal of functional importance. These results provide a starting point for structure of glycosylated human PEDF at 2.85 Å resolution. This future detailed structure͞function analyses into possible mecha- structure shows the location of a peptide previously identified as nisms of PEDF action that could lead to development of therapeu- having some of the neurotrophic properties of intact PEDF, but tics against uncontrolled angiogenesis. suggests that only restricted portions of this peptide are likely to be candidates for receptor interactions. It is unknown whether igment epithelium-derived factor (PEDF) is found as an the various functions of PEDF reside in different regions and Pextracellular component of the retinal interphotoreceptor involve binding to more than one receptor. Here it may be matrix (1), as well as in the vitreous and aqueous humors in the significant that the structure shows a strikingly asymmetric adult eye (2, 3). PEDF mRNA is, however, also found in most charge distribution that, in addition to better identifying a tissues (4). The Ϸ50-kDa protein is a member of the serpin proposed heparin-binding site that might play a role in PEDF superfamily, but does not inhibit proteinases (5). PEDF was localization, suggests another possible site of interaction with initially identified as a product secreted by cultured pigment cofactor proteins or other receptor molecules. epithelium cells from fetal human retina that possessed potent Methods neurotrophic activity in retinoblastoma cells (6). The PEDF gene was independently identified as one that is posttranscriptionally Protein Expression and Purification. Glycosylated human PEDF Ͼ was expressed and secreted by stably transfected BHK cells and and very markedly ( 100-fold) up-regulated (7) during G0 phase in young but not senescent cultured fibroblasts (8, 9), giving rise initially purified from the cell medium by ammonium sulfate to an alternative name of ‘‘early population doubling level fractionation and SP-Sepharose ion-exchange chromatography as described (3, 28). The protein sample was dialyzed against 50 cDNA’’ (EPC-1), suggesting additional roles in both the cell ⅐ ͞ cycle and senescence. mM Tris HCl, pH 8.0 20 mM NaCl and loaded onto a pre- Addition of PEDF at nanomolar concentrations induces a equilibrated Q-Sepharose column. PEDF was eluted with a 0.1–0.4 M NaCl gradient at a rate of 2 ml⅐minϪ1 during 60 min. neuronal phenotype in both cultured human retinoblastoma Y79 ͞ and Weri cells (10). PEDF promotes neuronal survival of the The purity of the PEDF sample was ascertained by SDS 12% cerebellar granule (11) and both survival and differentiation of PAGE. Glycosylation of the protein was checked by treatment of the denatured protein with N-glycosidase F followed by SDS͞ developing spinal motor neurons (12). It prevents death of cerebellar granule neurons (13), spinal motor neurons (14), and 12% PAGE. The sample was filtered through a 0.22- m mem- brane, concentrated to 17 mg⅐mlϪ1 by using a 10-kDa cutoff developing primary hippocampal neurons (15) caused by gluta- Ϫ mate cytotoxicity. It prevents hydrogen peroxide-induced apo- membrane, and stored at 80°C until needed. ptosis of retinal neurons (16), delays death of photoreceptors in BIOCHEMISTRY Crystallization. Crystallization conditions were identified in PEG- the mouse model of retinitis pigmentosa (17), supports both ion Hampton crystallization screen (29). Crystals were obtained normal development of the photoreceptor neurons and opsin at 18°C by using the hanging-drop vapor diffusion method, expression after removal of the retinal pigment epithelium (18), Ϫ mixing equal volumes of the protein (17 mg⅐ml 1) and the well and inhibits microglial growth (19). solution (0.2 M ammonium fluoride͞20% PEG 3,350, pH 6.20). Notwithstanding the various reports of the ability of PEDF to Rod-shaped crystals grew within a month to average dimensions affect the growth and differentiation of both retinoblastoma cells of 0.4 ϫ 0.15 ϫ 0.08 mm. The crystals belong to space group and normal neurons, perhaps the most exciting findings are the recent reports that PEDF is the most potent inhibitor of angiogenesis in the mammalian ocular compartment as well as a This paper was submitted directly (Track II) to the PNAS office. potent proliferation inhibitor in various cell types that acts Abbreviations: PEDF, pigment epithelium-derived factor; ␣1PI, ␣1-proteinase inhibitor. against a broad range of angiogenic͞proliferative inducers (20, Data deposition: The atomic coordinates and structure factors reported in this paper have 21), probably through an apoptotic mechanism (22). Neovascu- been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 1IMV). larization of the retina leads to ischemic retinopathies, such as ‡To whom reprint requests should be addressed. E-mail:[email protected]. proliferative diabetic retinopathy and age-related macular de- The publication costs of this article were defrayed in part by page charge payment. This generation (23), currently the leading causes of blindness in the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Western world, and the failure to exclude blood vessels from the §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.211268598 PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11131–11135 Downloaded by guest on October 3, 2021 ϭ ϭ ϭ P21212(a 176.17 Å; b 62.51 Å; and c 45.41 Å) with one Table 1. Data collection and refinement statistics Ϸ molecule per asymmetric unit, and 58% solvent content. Crystal Space group P21212 Data Collection, Structure Determination, and Refinement. Diffrac- Cell dimensions, Å a ϭ 176.2; b ϭ 62.5; c ϭ 45.4 tion data were collected at room temperature from three crystals Number of crystals 3 on a Rigaku RAXIS II detector, by using focused Cu K␣ Data collection radiation from a Rigaku RU-H2R rotating anode x-ray gener- Resolution limit, Å 100–2.85 ator at a power of 50 kV, 100 mA. The data were indexed with Wavelength, Å 1.514 DENZO, and scaled and reduced with SCALEPACK (30). The initial Reflections (observed͞unique) 152, 138͞11,241 phases were determined by molecular replacement using the Completeness͞last shell, % 91.2͞65.4 native structure of ␣ -proteinase inhibitor (␣ -PI; PDB accession 1 1 Rmerge (overall͞last shell), % 12.7͞37 code: 1QLP). The search model did not contain the reactive I͞I 8.5 center loop, loop 170, and the loop around residue 320 that Refinement connects s6A with s5A (loop 320). Molecular replacement Number of molecules in 1 calculations were performed with CRYSTALLOGRAPHY & NMR asymmetric unit SYSTEM (31). The rotation search gave a 6.8 peak, and the Resolution, Å 40.00–2.85 translation search gave a solution with a correlation coefficient Average B factor, Å2 31.4 of 28%. The oriented search model was then divided into several Number of reflections (work͞test) 10,075͞1,166 segments and refined as rigid bodies followed by cycles of Number of protein atoms 2,923 (375) simulated annealing giving an Rcryst of 41% (20–3.20 Å). Elec- (amino acids) tron-density maps were interpreted by using the program Number of solvent molecules 45 QUANTA (32). Regions of the model with poor density were Number of heterogen atoms 14 deleted and regions of insertions were identified. Ten percent of (carbohydrate) the data were randomly assigned to an R test for cross- free Rcryst (͉F͉ Ͼ 0) 18.8 validation (31). The model was progressively refined by using Rfree (͉F͉ Ͼ 0) 22.7 simulated annealing protocols, followed by energy minimization rms deviations from ideality and manual inspection and rebuilding. Resolution was increased Bond lengths, Å 0.007 stepwise. Once Rcryst dropped below 35%, bulk-solvent and Bond angles, ° 1.3 anisotropic B-factor corrections were introduced. Electron den- sity for missing loops as well as for the carbohydrate residue appeared strongly once Rcryst dropped below 28%. However, 15 amino acids at the N terminus and 8 amino acids in the reactive hydrophobic core is shown in Fig. 2. The N and C termini of the center loop were not visible in the final electron density maps.