Copper(II) Binding to the Human Doppel Protein May Mark Its Functional Diversity from the Prion Protein
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m404341200
ISSN1083-351X
AutoresGrazia M. Cereghetti, Alessandro Negro, Evi Vinck, Maria Lina Massimino, Maria Catia Sorgato, Sabine Van Doorslaer,
Tópico(s)Neurological diseases and metabolism
ResumoDoppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrPC) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrPC seems to be involved in Cu2+ recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrPC and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5–6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrPC, addition of Cu2+ to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrPC and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins. Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrPC) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrPC seems to be involved in Cu2+ recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrPC and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5–6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrPC, addition of Cu2+ to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrPC and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins. Doppel (Dpl, downstream prion protein-like gene or German for "double") is the first described homologue (1Moore R.C. Lee I.Y. Silverman G.L. Harrison P.M. Strome R. Heinrich C. Karunaratne A. Pasternak S.H. Chishti M.A. Liang Y. Mastrangelo P. Wang K. Smit A.F.A. Katamine S. Carlson G.A. Cohen F.E. Prusiner S.B. Melton D.W. Tremblay P. Hood L.E. Westaway D. J. Mol. Biol. 1999; 292: 797-817Crossref PubMed Scopus (473) Google Scholar) of the prion protein, PrPC. 1The abbreviations used are: PrP, prion protein; PrPC, cellular PrP; MOPS, 3-(N-morpholino)propanesulfonic acid; CNS, central nervous system; bPrP, bovine PrP; GFP, green fluorescent protein; NEM, N-ethylmorpholine; CHO, Chinese hamster ovary; TSEs, transmissible spongiform encephalopathies; BSE, bovine spongiform encephalopathy; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; GPI, glycosylphosphatidylinositol; hu, human; m, mouse.1The abbreviations used are: PrP, prion protein; PrPC, cellular PrP; MOPS, 3-(N-morpholino)propanesulfonic acid; CNS, central nervous system; bPrP, bovine PrP; GFP, green fluorescent protein; NEM, N-ethylmorpholine; CHO, Chinese hamster ovary; TSEs, transmissible spongiform encephalopathies; BSE, bovine spongiform encephalopathy; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; GPI, glycosylphosphatidylinositol; hu, human; m, mouse. PrPC is a cellular glycoprotein of still enigmatic functions and is expressed in higher amounts in the central nervous system (CNS). If present as a conformational isoform called PrPSc, it causes a class of diseases known as transmissible spongiform encephalopathies (TSEs) or prion diseases (2Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4094) Google Scholar, 3Prusiner S.B. Brain Pathol. 1998; 8: 499-513Crossref PubMed Scopus (311) Google Scholar). PrPSc seems to be the main, if not the unique, component of the infectious agent of TSEs, the prion (3Prusiner S.B. Brain Pathol. 1998; 8: 499-513Crossref PubMed Scopus (311) Google Scholar). TSEs, such as bovine spongiform encephalopathy (BSE) in cattle, CreutzfeldtJakob disease, Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia in humans, are fatal, progressive, and neurodegenerative disorders of genetic, sporadic, or infectious origin. After the BSE epidemics in Great Britain, the most alarming TSE member is the new variant CreutzfeldtJakob disease, probably caused by the consumption of BSE-contaminated meat products (4Collinge J. Sidle K.C. Meads J. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1589) Google Scholar, 5Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (857) Google Scholar). Dpl can be regarded as an N-truncated form of PrPC. It shares 26% sequence homology and an almost superimposable three-dimensional fold, characterized by three α-helices and two short antiparallel β-sheets, with the structured C-terminal domain of PrPC (1Moore R.C. Lee I.Y. Silverman G.L. Harrison P.M. Strome R. Heinrich C. Karunaratne A. Pasternak S.H. Chishti M.A. Liang Y. Mastrangelo P. Wang K. Smit A.F.A. Katamine S. Carlson G.A. Cohen F.E. Prusiner S.B. Melton D.W. Tremblay P. Hood L.E. Westaway D. J. Mol. Biol. 1999; 292: 797-817Crossref PubMed Scopus (473) Google Scholar, 6Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (664) Google Scholar, 7Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar, 8Mo H. Moore R.C. Cohen F.E. Westaway D. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2352-2357Crossref PubMed Scopus (141) Google Scholar, 9Luhrs T. Riek R. Guntert P. Wuthrich K. J. Mol. Biol. 2003; 326: 1549-1557Crossref PubMed Scopus (64) Google Scholar). The structure of Dpl is, however, stabilized by the presence of an additional disulfide bridge, which may also explain the incapacity of Dpl to convert into a PrPSc-like pathogenic conformer (10Rossi D. Cozzio A. Flechsig E. Klein M.A. Rulicke T. Aguzzi A. Weissmann C. EMBO J. 2001; 20: 694-702Crossref PubMed Scopus (224) Google Scholar). Dpl is not required for prion replication (11Moore R.C. Mastrangelo P. Bouzamondo E. Heinrich C. Legname G. Prusiner S.B. Hood L. Westaway D. DeArmond S.J. Tremblay P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15288-15293Crossref PubMed Scopus (132) Google Scholar), and it is mainly expressed in heart and spermatozoa (12Behrens A. Genoud N. Naumann H. Rulicke T. Janett F. Heppner F.L. Ledermann B. Aguzzi A. EMBO J. 2002; 21: 3652-3658Crossref PubMed Scopus (135) Google Scholar, 13Peoc'h K. Serres C. Frobert Y. Martin C. Lehmann S. Chasseigneaux S. Sazdovitch V. Grassi J. Jouannet P. Launay J.M. Laplanche J.L. J. Biol. Chem. 2002; 277: 43071-43078Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), although only transiently in epithelial cells of the CNS during the 1st week after birth (14Li A. Sakaguchi S. Shigematsu K. Atarashi R. Roy B.C. Nakaoke R. Arima K. Okimura N. Kopacek J. Katamine S. Am. J. Pathol. 2000; 157: 1447-1452Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Nevertheless, the ectopic expression of Dpl in the CNS of PrP0/0 mice results in ataxia due to loss of cerebellar granules and Purkinje cells. As the healthy phenotype is rescued by reintroducing the wild-type PrP transgene (1Moore R.C. Lee I.Y. Silverman G.L. Harrison P.M. Strome R. Heinrich C. Karunaratne A. Pasternak S.H. Chishti M.A. Liang Y. Mastrangelo P. Wang K. Smit A.F.A. Katamine S. Carlson G.A. Cohen F.E. Prusiner S.B. Melton D.W. Tremblay P. Hood L.E. Westaway D. J. Mol. Biol. 1999; 292: 797-817Crossref PubMed Scopus (473) Google Scholar, 11Moore R.C. Mastrangelo P. Bouzamondo E. Heinrich C. Legname G. Prusiner S.B. Hood L. Westaway D. DeArmond S.J. Tremblay P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15288-15293Crossref PubMed Scopus (132) Google Scholar), the two proteins may have related, albeit opposite, functions in the CNS. PrPC and Dpl are both anchored to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) molecule. This peculiar surface location, together with the ability of PrPC to bind copper(II) (Cu(II)) ions, particularly within the PHGGGWGQ consensus sequence of the N-terminal octapeptide repeats (15Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar, 16Stockel J. Safar J. Wallace A.C. Cohen F.E. Prusiner S.B. Biochemistry. 1998; 37: 7185-7193Crossref PubMed Scopus (493) Google Scholar, 17Viles J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar, 18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar)), has led to the hypothesis that PrPC acts as a Cu(II)-removing protein from synaptic clefts (19Herms J. Tings T. Gall S. Madlung A. Giese A. Siebert H. Schurmann P. Windl O. Brose N. Kretzschmar H. J. Neurosci. 1999; 19: 8866-8875Crossref PubMed Google Scholar). An involvement of PrPC in copper metabolism is also supported by the finding that the ion induces internalization of the protein in cell culture systems (20Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 21Lee K.S. Magalhaes A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). Recently, we have shown also that the structured C-terminal domain of the protein is able to bind Cu(II) with high specificity, opening the possibility that Cu(II) binding to this region may have an important functional role (18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Van Doorslaer S. Cereghetti G.M. Glockshuber R. Schweiger A. J. Phys. Chem. 2001; 105: 1631-1639Crossref Scopus (74) Google Scholar, 23Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2003; 84: 1985-1997Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), perhaps related to the proposed antioxidant, superoxide dismutase-like activity of the protein (24Brown D.R. Wong B.S. Hafiz F. Clive C. Haswell S.J. Jones I.M. Biochem. J. 1999; 344: 1-5Crossref PubMed Scopus (494) Google Scholar). Because PrPC and Dpl have highly similar globular folds and contain histidine residues that appear to be good candidates for Cu(II) binding, we argued that Dpl could also bind the ion (18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Indeed, during the accomplishment of this work, Westaway and co-workers (25Qin K. Coomaraswamy J. Mastrangelo P. Yang Y. Lugowski S. Petromilli C. Prusiner S.B. Fraser P.E. Goldberg J.M. Chakrabartty A. Westaway D. J. Biol. Chem. 2003; 278: 8888-8896Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) have shown by mass spectrometry and fluorescence measurements that recombinant mouse Dpl (mDpl) contains a specific Cu(II)-binding site at neutral pH, which probably involves histidine residue 131. Here, we report on the investigation of the Cu(II) binding behavior of recombinant human Dpl, huDpl-(28–152), by EPR and fluorescence spectroscopy. Assays were carried out in the pH range 3–8 not only to clarify fully the binding characteristics of the ion but also to probe copper binding capacities at those pH values that may be experienced physiologically by a protein (pH 4–6 and 7.4). This study shows that Cu(II) binding to Dpl starts only at pH 4 and that at acidic pH values two types of Cu(II)-binding sites are detected as follows: one is visible at pH 4, and the other is observed in the pH range 5–6. Fluorescence quenching measurements show that none of these sites involve a tryptophan residue. At pH 7.4, both specific and nonspecific Cu(II)-binding sites are identified. Moreover, we tested at this pH value the influence of copper on the secondary structure of Dpl using circular dichroism spectroscopy. Finally, in order to get insight into the functional impact of Cu(II) binding to Dpl, we analyzed whether the metal induces internalization of the protein in cell cultures, as was observed previously for PrPC. We found that Cu2+ addition does not induce massive internalization of Dpl, suggesting that Cu(II) binding to Dpl and PrPC may have different functional implications. The sequence encoding human Dpl-(28–152) (huDpl-(28–152)) was amplified by PCR using human placenta DNA as template and the following primers: 5′-AAGAATTCAGCCCCTCTCCAACCAAAACTCGCAA-3′ and 5′-CCGGATCCAGGGGCATCAAGCACAGAATCAAGTG-3′. The amplified product was cloned in the pRSET A plasmid (Invitrogen) between BamHI and EcoRI restriction sites, whereby the 28–152-residue amino acid sequence of Dpl was N-terminally fused to a polyhistidine tag and to a thrombin cleavage site. The latter consists of residue 28 of huDpl and an Ile-Ser substitution at position 29. The plasmid was verified by double-stranded DNA sequencing (ABI Prism kit, Applied Biosystems, Foster City, CA). Expression was carried out in BL-21(D3) Escherichia coli cells grown at 37 °C on Luria broth medium containing 100 mg/liter ampicillin. When cells reached an absorbance of 0.6 at 600 nm, Dpl expression was induced by adding 1 mm isopropyl-β-d-thiogalactoside (Sigma) (4 h, 37 °C). Cells were harvested by centrifugation. huDpl-(28–152) was purified as described by Negro et al. (26Negro A. Meggio F. Bertoli A. Battistutta R. Sorgato M.C. Pinna L.A. Biochem. Biophys. Res. Commun. 2000; 271: 337-341Crossref PubMed Scopus (33) Google Scholar) with the following minor modifications. After solubilization from inclusion bodies (6 m guanidinium chloride, pH 8.0), the protein was immobilized on a metal affinity Ni2+-nitrilotriacetate-agarose column (Qiagen GmbH, Hilden, Germany), refolded on the column in the presence of refolding buffer (20 mm Tris/HCl, pH 8.0, 50 mm NaCl), detached from the histidine tag by addition of thrombin (10 units, Sigma), and eluted from the column (50 mm sodium acetate, pH 5.8). Purified huDpl was dialyzed twice (24 h) against 50 mm ammonium acetate, pH 4.5, and then against Millipore water (20 h). The protein was stored at –80 °C. Analysis by MALDI-MS of the intact protein and of its proteolytic digests, under oxidized or reduced conditions, proved that Dpl has the expected mass (calculated average Mr of oxidized Dpl, including the Ile-Ser substitution at position 29, 14,312.05; observed Mr, 14,312.5), with two intramolecular disulfide bonds mapped at the predicted positions of the human sequence (between cysteine residues 94–145 and 108–140) (1Moore R.C. Lee I.Y. Silverman G.L. Harrison P.M. Strome R. Heinrich C. Karunaratne A. Pasternak S.H. Chishti M.A. Liang Y. Mastrangelo P. Wang K. Smit A.F.A. Katamine S. Carlson G.A. Cohen F.E. Prusiner S.B. Melton D.W. Tremblay P. Hood L.E. Westaway D. J. Mol. Biol. 1999; 292: 797-817Crossref PubMed Scopus (473) Google Scholar). Sample Preparation for EPR and Fluorescence Spectroscopy Measurements—The following buffers were used (all at 10 mm concentration): formic acid/sodium hydroxide, pH 3 and 4; sodium acetate/hydrochloride, pH 5.0; sodium cacodylate/hydrochloride, pH 6; and MOPS/sodium hydroxide pH 7.4 and 8. For the EPR measurements at the different pH values, 1–7 molar eq of CuCl2 were added to a stock solution of recombinant huDpl to obtain a final protein concentration of 100 μm (final sample volume 70–100 μl). Alternatively, the metal-free protein was first dialyzed against the desired buffer solution containing 50 μm CuCl2, and afterward against the same buffer without Cu2+, to remove free Cu2+. To improve the spectral quality, 10% (v/v) glycerol was added as a cryoprotectant. Control measurements were performed to ascertain whether glycerol influenced EPR parameters. Because glycerol changed the Cu(II) buffer signal only at pH 5, it was omitted from all measurements at this pH. For fluorescence spectroscopy measurements, stock huDpl-(28–152) solutions were diluted to a final protein concentration of 0.7 μm, using the above described buffer solutions as well as HEPES and N-ethylmorpholine (NEM)-KCl buffers, both at 10 mm concentration, pH 7.4. By using a concentrated CuCl2 solution to minimize the sample dilution (lower than 2%), 3–20 molar eq of CuCl2 were added to the 0.7 μm solution of huDpl. Fluorescence quenching was also studied in the presence of 4 molar eq of ZnCl2, MgCl2, or CaCl2. Continuous Wave EPR Spectroscopy—EPR spectra were recorded on a Bruker ESP300E spectrometer (microwave frequency 9.43 GHz) equipped with a gas-flow cryogenic system, allowing operation from room temperature down to 2.5 K. All spectra were recorded with a microwave power of 10 milliwatts, a modulation frequency of 100 kHz, and a modulation amplitude of 0.5 millitesla. All experiments were performed at 90 K. The magnetic field was measured with a Bruker ER 035M NMR gaussmeter and was calibrated by using a sample of diphenylpicrylhydrazyl. The EPR parameters were determined by simulation of the EPR spectra using the EasySpin program (www.esr.ethz.ch), whereby the contribution of both copper isotopes (63Cu and 65Cu) were taken into account. Circular Dichroism Spectroscopy—Far-UV circular dichroism spectra were measured at 25 °C on a Jasco J-715 spectropolarimeter in 0.1-cm quartz cuvettes, accumulated eight times, and corrected for the background. Protein samples of 10 μm huDpl in 10 mm MOPS/NaOH buffer, pH 7.4, in the presence or absence of 1 or 2 molar eq of CuCl2 were analyzed. Fluorescence Spectroscopy—Steady-state fluorescence spectra were recorded on a Cary spectrophotometer. Emission spectra were collected from 290 to 500 nm (λex = 280 nm, 0.5 nm/s, bandpass 5 nm for excitation and emission). Fluorescence intensities were integrated from 290 to 480 nm after subtraction of the background. The incubation time before fluorescence measurements was varied to allow protein-Cu(II) interactions. The protein was incubated at room temperature or at 4 °C. For each experimental condition analyzed, a control sample without Cu2+ was measured to correct for degradation of the protein during the incubation time. Plasmid Construction—cDNAs coding for the eukaryotic expression of human PrP and human Dpl fused to the green fluorescent protein (GFP) (huPrP-GFP and huDpl-GFP) were constructed as described previously (27Massimino M.L. Ballarin C. Bertoli A. Casonato S. Genovesi S. Negro A. Sorgato M.C. Int. J. Biochem. Cell Biol. 2004; 36: 2026-2041Crossref Scopus (23) Google Scholar). The plasmid encoding the chimeric GFP-GPIPrP protein was obtained from the plasmid pGFP-bPrP (encoding for bovine PrP (bPrP) fused to GFP) (28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar), after deletion of bPrP sequence 43–221, using inverse PCR and the following primers: 5′-TCCAGATCTGAGTCCGGACTTGTACAGCTC-3′ and 5′-AGAAGATCTCAGGCTTATTACCAAGGGGGGC-3′. The resulting construct codes for GFP linked to bPrP leader sequence 1–42, and to bPrP sequence 222–256 (which includes the attachment signal for GPI), at the N and C terminus, respectively. Cell Culture—Wild-type or stably transfected CHO cells were maintained at 37 °C in Ham's F-12 medium, 10% fetal bovine serum (Invitrogen), 2 mm l-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen.), in 75-cm2 culture bottles in a 5% CO2 atmosphere. The medium was changed every 2–3 days. The transient transfection of wild-type cells was performed 1 day after plating cells on 24-mm coverslips, using the liposome-mediated method (LipofectAMINE Plus, Invitrogen) and Opti-MEM medium, following the manufacturer's instructions. 1 μg of plasmid was added to each well (105 cells/well). To optimize protein expression, 4 h after transfection the medium was changed, and cells were kept at 30 °C for 72 h before starting the endocytosis experiment. CHO cells stably expressing huPrP- or huDpl-GFP were established as described by Negro et al. (28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar). Before use, cells were plated on 24-mm coverslips (105 cells/well), grown at 37 °C for 24 h, and then kept for the next 24 h at 30 °C. Fluorescence Imaging—Copper-induced protein internalization was followed in single live cells by means of the GFP fluorescence (excitation at 488 nm; emission at 509 nm), using a Zeiss Axiovert 100 microscope equipped with a 16-bit digital CCD videocamera (Micromax, Princeton Instruments, Trenton, NJ). Images were taken at 5-min intervals, before and after the addition of 200 or 500 μm Cu(II) acetate (pH 7.4, room temperature), and analyzed using the Metafluor or Metamorph software (Universal Imaging). Alternatively, after 40 min of incubation at 37 °C, in Ham's F-12 culture medium with or without 500 μm Cu(II) acetate, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (4 °C, 30 min) and then incubated in chilled methanol (5 min, –20 °C). After washing with phosphate-buffered saline, coverslips were mounted in glycerol for observation under the fluorescence microscope. EPR Experiments—The EPR spectra of huDpl-(28–152) in the presence of copper (Figs. 1,2,3 to Fig. 4) are typical for type 2 protein-Cu(II) complexes (axial g matrix, copper hyperfine splitting, A∥ >400 MHz) (29Messerschmidt A. Struct. Bonding. 1998; 90: 37-68Crossref Google Scholar). Type 2 complexes are largely square planar with a possible fifth weak coordination. For type 2 Cu(II) complexes, (g∥, A∥) values correlate with the type of the equatorially coordinating atoms (30Peisach J. Blumberg W.E. Arch. Biochem. Biophys. 1974; 165: 691-708Crossref PubMed Scopus (1191) Google Scholar). Unfortunately, the g∥ and A∥ values depend also on other parameters (such as the charge of the surrounding ligands and a possible fifth ligand). This hampers a clear-cut determination of the first coordination sphere of the Cu(II) on the basis of the CW-EPR data alone. Nevertheless, the g∥ and A∥ values can be used to get a first idea about the coordinating atoms and are very useful in the comparison of the Cu(II) binding of prion and doppel proteins.Fig. 2X-band EPR spectra of huDpl-(28–152) in the presence of Cu2+ at pH 8. EPR spectra of 0.1 mm huDpl-(28–152) with 1 molar eq Cu2+ added (a) and 0.1 mm huDpl-(28–152) with 3 molar eq Cu2+ added (b).View Large Image Figure ViewerDownload (PPT)Fig. 3X-band EPR spectra of huDpl-(28–152) in the presence of Cu2+ at pH 4. EPR spectra of the pH 4 formate buffer solution with 0.3 mm CuCl2 (a) and 0.1 mm huDpl-(28–152) with 2 molar eq Cu2+ added (b).View Large Image Figure ViewerDownload (PPT)Fig. 4X-band EPR spectra of huDpl-(28–152) in the presence of Cu2+ at pH 5 and 6. EPR spectra of the pH 5 acetate buffer solution with 0.5 mm CuCl2 (a), 0.1 mm huDpl-(28–152) with 2 molar eq Cu2+ added at pH 5 (b), 0.1 mm huDpl-(28–152) with 2 molar eq Cu2+ added at pH 6 (c), and the pH 6 cacodylate buffer solution with 0.4 mm CuCl2 (d).View Large Image Figure ViewerDownload (PPT) Fig. 1a shows the control EPR spectrum of the MOPS buffer, pH 7.4, with 2 mm Cu2+. At pH ≥ 7, aquo-Cu(II) has only low solubility and precipitates largely as EPR silent [Cu(OH)2]n,so that no Cu(II) signal can be observed in the control. The signal indicated by *, in Fig. 1a, is a cavity signal. Fig. 1b shows the EPR spectrum of 100 μm huDpl-(28–152) dialyzed against a buffer solution containing 50 μm CuCl2 with subsequent dialysis against the same buffer without Cu2+ in order to remove unbound Cu2+. An EPR spectrum typical for a type 2 Cu(II) complex is visible (EPR parameters given in Table I, complex D3). The spectrum is analogous to the one obtained after addition of 1 molar eq of Cu2+ to 100 μm of the protein, without dialysis at pH 7.4 (Fig. 1c) and at pH 8 (Fig. 2a). After addition of more equivalents of Cu2+, a second Cu(II) complex becomes dominant (Fig. 1, d–f, Fig. 2b, and complex D4 in Table I). Because this second complex does not appear in the dialyzed samples (Fig. 1b) and appears only after addition of an excess of copper, the corresponding binding site(s) will have a low binding affinity for Cu2+ and is probably of no biological relevance. The increase of the EPR intensity of the D4 contribution with increasing Cu(II) concentration indicates that more than one nonspecific complex with similar structure is formed per Dpl protein (Fig. 1).Table IEPR parameters of the type 2 Cu(II) complexes observed in copper-containing huDpl-(28–152)Complexg∥ (± 0.005)g⊥ (± 0.005)A∥/MHz (±10)A⊥/MHz (±20)pHBufferRef.huDpl-(28-152)NBNBNBNB3FThis workmPrP-(23-231)P12.3322.06845212F18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarP22.2952.06845720FhuDpl-(28-152)D12.3202.065490304FThis workmPrP-(23-231)P12.3322.06845212F18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarP22.2952.06845720F18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarhuDpl-(28-152)D22.2802.058522305AThis workmPrP-(23-231)P12.3322.06845212A18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarP22.2952.06845720A18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarhuDpl-(28-152)D22.2802.058522306CThis workmPrP-(23-231)P12.3322.06845212C18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarP22.2952.06845720C18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarmPrP-(58-91)P32.2702.05552050C or N18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 32Aronoff-Spencer E. Burns C.S. Avdievich N.I. Gerfen G.J. Peisach J. Antholine W.E. Ball H.L. Cohen F.E. Prusiner S.B. Millhauser G.L. Biochemistry. 2000; 39: 13760-13771Crossref PubMed Scopus (326) Google ScholarhuDpl-(28-152)D32.2052.045592607-8MThis workD42.2602.05556030MThis workmPrP-(23-231)P42.2952.06845720M18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarP32.2302.05549550M18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google ScholarmPrP-(58-91)P52.2702.05552050M or N18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Burns C.S. Aronoff-Spencer E. Legname G. Prusiner S.B. Antholine W.E. Gerfen G.J. Peisach J. Millhauser G.L. Biochemistry. 2003; 42: 6794-6803Crossref PubMed Scopus (261) Google ScholarP32.2302.05549550M or N18Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Burns C.S. Aronoff-Spencer E. Legname G. Prusiner S.B. Antholine W.E. Gerfen G.J. Peisach J. Millhauser G.L. Biochemistry. 2003; 42: 6794-6803Crossref PubMed Scopus (261) Google ScholarPrP-(90-101)P62.21588N33Burns C.S. Aronoff-Spencer E. Legname G. Prusiner S.B. Antholine W.E. Gerfen G.J. Peisach J. Millhauser G.L. Biochemis
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