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Highly Accelerated Self-Assembly and Fibrillation of Prion Peptides on Solid Surfaces Sook Hee Ku and Chan Beum Park* Institute for the BioCentury and Department of Materials Science and Engineering, Korea AdVanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon, Republic of Korea ReceiVed September 6, 2008. ReVised Manuscript ReceiVed NoVember 5, 2008 The conformational change of cellular prion protein (PrPC) to its infectious isoform (PrPSc) is a hallmark of prion diseases. We have developed a novel solid surface-based system for efficient prion fibrillation in vitro by immobilizing prion peptides onto a chemically activated solid surface. The self-assembly of prion peptides into fibrils was more highly accelerated on the solid surface than in solution after 72 h of incubation at 37 °C. According to our observation using ex situ atomic force microscopy, fibrils were over 200 nm long and 5-8 nm in diameter. Amyloid-like properties of fibrils self-assembled on the solid surface were confirmed by multiple analyses with circular dichroism and amyloidspecific dyes such as Congo red and thioflavin T. The fibril formation of prion peptides was substantially affected by the incubation temperature, and preformed fibrils disassembled after additional heat treatment at 100 °C. The solid surface-based prion fibrillation system developed in the present work may become a useful tool for the in vitro study of prion aggregation. The adoption of this system will allow the efficient investigation of environmental factors and inhibitor screening.
Introduction Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the structural transition of prion protein. In these diseases, abnormal isoforms (PrPSc) of cellular prion (PrPC) accumulate in neuronal tissues.1 PrPC changes into an insoluble, protease-resistant PrPSc form having a cross β sheet rich conformation through a posttranslational process, which further self-assembles into amyloid fibrils. Although the cellular mechanism of amyloid formation remains unclear and controversial, PrPSc is believed to induce prion diseases.2,3 Until now, many researchers have tried to explore in vitro fibrillation of prion protein by dissolving the protein or its fragments in aqueous solutions;4-6 however, the conventional “solution-based” method exhibited many problems (e.g., extremely slow fibrillation and the formation of a highly heterogeneous mixture of aggregates). Thus, a new model system for in vitro study of prion fibrillation is critically needed to overcome these drawbacks. Here we first report the development of a solid surface-based in vitro model system for prion fibrillation through the immobilization of prion peptide on a chemically activated solid surface. According to the literature, prions are natively glycosylphosphatidylinositol (GPI)-anchored surface proteins1 (Figure 1A) and are located in caveolae that are the sphingolipid/ cholesterol-rich regions of the cell membrane, like other GPIanchored proteins. Several studies showed that the conversion * To whom correspondence should be addressed. Tel: +82-42-350-3340. Fax: +82-42-350-3310. E-mail:
[email protected]. (1) Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13363–13383. (2) Telling, G. C.; Parchi, P.; DeArmond, S. J.; Cortelli, P.; Montagna, P.; Gabizon, R.; Mastrianni, J.; Lugaresi, E.; Gambetti, P.; Prusiner, S. B. Science 1996, 274, 2079–2082. (3) Prusiner, S. B. Science 1997, 278, 245–251. (4) Tagliavini, F.; Prelli, F.; Verga, L.; Giaccone, G.; Sarma, R.; Gorevic, P.; Ghetti, B.; Passerini, F.; Ghibaudi, E.; Forloni, G.; Salmona, M.; Bugiani, O.; Frangione, B. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9678–9682. (5) Salmona, M.; Malesani, P.; De Gioia, L.; Gorla, S.; Bruschi, M.; Molinari, A.; Della Vedova, F.; Pedrotti, B.; Marrari, M. A.; Awan, T.; Bugiani, O.; Forloni, G.; Tagliavini, F. Biochem. J. 1999, 342, 207–214. (6) Petty, S. A.; Adalsteinsson, T.; Decatur, S. M. Biochemistry 2005, 44, 4720–4726.
of prion protein probably occurs in caveolae.7-9 Recently, surfaceimmobilized prion peptides have been used to identify recognition elements or the structural rearrangement portion of prion protein.10,11 Furthermore, the fibrillation rate of other amyloidogenic proteins such as Alzheimer’s β-amyloid, insulin, and the immunoglobulin light-chain variable domain was enhanced in the presence of a solid surface rather than in the solution phase.12-14 Considering these aspects, we suggest that a solid surface-based fibrillation of prion should be a more attractive route for the study of prion self-assembly and fibril formation in vitro. The model prion peptide used in the present study corresponds to residues 106-126 of human prion protein (Figure 1A). The peptide had been widely used as an appropriate model for the in vitro study of prion amyloid formation and neurotoxicity because of its similar physicochemical properties to PrPSc.4,15-19 According to our observation using ex situ atomic force microscopy (AFM), the self-assembly of prion peptides into fibrils was more highly accelerated on the solid surface than in the solution phase. We further analyzed the secondary structure of (7) Vey, M.; Pilkuhn, S.; Wille, H.; Nixon, R.; De Armond, S. J.; Smart, E. J.; Anderson, R. G. W.; Taraboulos, A.; Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14945–14949. (8) Taraboulos, A.; Scott, M.; Semenov, A.; Avraham, D.; Lsazlo, L.; Prusiner, S. B. J. Cell Biol. 1995, 129, 121–132. (9) Naslavsky, N.; Shmeeda, H.; Friedlander, G.; Yanai, A.; Futerman, A. H.; Barenholz, Y.; Taraboulos, A. J. Biol. Chem. 1999, 274, 20763–20771. (10) Tessier, P. M.; Lindquist, S. Nature 2007, 447, 556–562. (11) Leclerc, E.; Peretz, D.; Ball, H.; Sakurai, H.; Legname, G.; Serban, A.; Prusiner, S. B.; Burton, D. R.; Williamson, R. A. EMBO J. 2001, 20, 1547–1554. (12) Ha, C.; Park, C. B. Langmuir 2006, 22, 6977–6985. (13) Ha, C.; Park, C. B. Biotechnol. Bioeng. 2005, 90, 848–855. (14) Zhu, M.; Souillac, P. O.; Ionescu-Zanetti, C.; Carter, S. A.; Fink, A. L. J. Biol. Chem. 2002, 277, 50914–50922. (15) De Gioia, L.; Selvaggini, C.; Ghibaudi, E.; Diomede, L.; Bugiani, O.; Forloni, G.; Tagliavini, F.; Salmona, M. J. Biol. Chem. 1994, 269, 7859–7862. (16) Selvaggini, C.; De Gioia, L.; Gantu, L.; Ghibaudi, E.; Diomede, L.; Passerini, F.; Forloni, G.; Bugiani, O.; Tagliavini, F.; Salmona, M. Biochem. Biophys. Res. Commun. 1993, 194, 1380–1386. (17) Rymer, D. L.; Good, T. A. J. Neurochem. 2000, 75, 2536–2545. (18) Forloni, G.; Angeretti, N.; Ghiesa, R.; Monzani, E.; Salmona, M.; Bugiani, O.; Tagliavini, F. Nature 1993, 362, 543–546. (19) Brown, D. R.; Pitschke, M.; Riesner, D.; Kretzschmar, H. A. Nerurosci. Res. Commun. 1998, 23, 119–128.
10.1021/la802931k CCC: $40.75 2008 American Chemical Society Published on Web 11/20/2008
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Figure 1. (A) Bar diagram of human prion protein. Prion is a natively GPI-anchored surface protein and contains two sheets and three helices in its native state. PrP106-126 was used as a model peptide in the present study. (B) Representative AFM images of fibrils formed in the solution phase and on a solid surface. The PrP106-126 solution (100 µM) was incubated at 37 °C for 72 h with or without the peptide-immobilized surface. The size of each AFM image is 2 µm × 2 µm.
fibrils formed on the solid surface by circular dichroism (CD) together with analysis using amyloid-specific dyes such as Congo red (CR) and thioflavin T (ThT). Using our solid-based in vitro system, we explored the effect of high temperature on the formation and stability of prion fibrils on solid surfaces.
Experimental Section Chemicals. PrP106-126 was custom-synthesized by Peptron Inc. (Daejeon, Korea). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), (3aminopropyl)-triethoxysilane (APTS), N,N′-disuccinimidyl carbonate (DSC), ethanolamine, 6-mercaptohexanoic acid (MHA), 11-mercaptoundecanoic acid (MUA), 16-mercaptohexadecanoic acid (MHDA), poly-L-lysine (PLL), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), succinic anhydride, dimethylformamide (DMF), Congo red (CR), and thioflavin T (ThT) were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile was purchased from Junsei Chemical Co. (Tokyo, Japan), and dextran amine (500 kDa) was purchased from Invitrogen (Carlsbad, CA). Preparation of PrP106-126 Solution. A stock solution of PrP106-126 was prepared as described previously.20 Briefly, lyophilized peptide was dissolved in HFIP and sonicated in a water bath for 3 min, and then aliquots were transferred to microcentrifuge tubes. The HFIP solvent was evaporated in a vacuum desiccator and stored at -20 °C. Prior to use, PrP106-126 peptide was dissolved in 200 mM acetate buffer (pH 5.5, 150 mM NaCl) containing 50% (v/v) acetonitrile at a desired concentration. Prion Peptide Fibrillation on N-Hydroxysuccinimide (NHS)Activated Glass Surfaces. We prepared NHS-activated glass surfaces as described earlier.12,13 Glass slides were cleaned with piranha solution (70% H2SO4/30% H2O2, 7:3 v/v) for 15 min at 60 °C and extensively washed with water. The slides were treated with 3% APTS in ethanol/water (95:5 v/v) for 1 h at room temperature and cured for additional hour at 110 °C. For NHS activation, the slides were incubated in 20 mM DSC solution in 50 mM sodium bicarbonate buffer (pH 8.5) for 3 h. After washing with water and drying with (20) Kanapathipillai, M.; Ku, S. H.; Girigoswami, K.; Park, C. B. Biochem. Biophys. Res. Commun. 2008, 365, 808–813.
N2, PrP106-126 peptide was immobilized on the glass for 10 min. Remaining functionalities were blocked with 1 M ethanolamine solution for 15 min and then washed with water. For prion peptide fibrillation, the slides were incubated in PrP106-126 solution at 37 °C. Prion Peptide Fibrillation on Various Self-Assembled Monolayer Surfaces. Gold-coated silicon wafers were cleaned by sonication with ethanol, followed by treatment with piranha solution. After rigorous washing, the wafers were dipped in 10 mM MHA, MUA, or MHDA solution in ethanol for more than 12 h, sonicated for 15 min, and washed with ethanol. The NHS functionality was obtained by treatment with an aqueous mixture of 0.1 M EDC and 0.025 M NHS for 10 min. After the immobilization of 100 µM PrP106-126 peptide, the wafers were incubated in the peptide solution at 37 °C for 72 h. Dextran or PLL surfaces were prepared by treatment with 0.1 mg/mL dextran amine (500 kDa) or PLL in 10 mM phosphate buffer (pH 7.0) on NHS-activated MUA surfaces for 1 h. The amine groups of dextran or PLL were carboxylated with 0.1 M succinic anhydride in DMF for 4 h and then washed with water. After treatment with a mixture of 0.4 M EDC and 0.1 M NHS, PrP106-126 peptide (100 µM) was covalently bonded, remaining functional groups were blocked with ethanolamine, and then templates were further incubated in PrP106-126 solution. Ex Situ Atomic Force Microscopy. The morphology of prion amyloid fibrils was observed by ex situ AFM. For the AFM analysis, a 100 µM PrP106-126 solution was used for the immobilization of peptide on an NHS-activated glass slide. The slide was incubated in 100 µM peptide solution at 37 °C for 0, 24, 48, or 72 h, respectively. The sample surface was scanned using a Nanoscope IIID Multimode AFM (Digital Instruments Inc.) under ambient conditions. AFM analysis was carried out in tapping mode under the following conditions: scan rate, 1.5 Hz; “E” scanner; NCHR silicon cantilever (Nanosensors Inc.); resonance frequency range of cantilevers, 250-350 kHz; and number of pixels, 512 × 512. Representative images were selected from at least five spots over the entire surface. Circular Dichroism (CD) Analysis. For the penetration of UV light, quartz plates were used instead of glass slides during CD measurements. Samples for CD analysis were prepared by the immobilization of PrP106-126 on quartz plates, followed by further
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Figure 2. Representative AFM images and section analysis of PrP106-126 fibrils formed on a synthetic template. The template was prepared by immobilizing PrP106-126 (100 µM) on an NHS-activated glass slide and then incubating in PrP106-126 solution (100 µM) at 37 °C for 0, 24, 48, and 72 h. The size of each AFM image is 2 µm × 2 µm.
incubation in a fresh PrP106-126 solution (800 µM) at 37 °C. The CD spectrum of each sample was obtained with a J-810 instrument (Jasco Co.) at a scan speed of 500 nm/min and a step resolution of 2 nm from 186 to 245 nm. Congo Red (CR) Binding. Prion fibrils formed on the surface were quantified by CR binding. For CR binding, the surfaces of glass slides were immobilized with PrP106-126 and then they were incubated in fresh PrP106-126 solution (200 µM) for the fibrillation on the glass surface. After incubation, each slide sample was dipped for 10 min in a 24-well plate containing 25 µM CR solution (20 mM Tris-HCl, pH 8.0, 10 v/v% ethanol). After washing with deionized water, slide samples were placed in the Tris-HCl buffer solution, followed by the measurement of absorbance at 535 nm using Victor 3 microplate reader (Perkin-Elmer). Thioflavin T (ThT)-Induced Fluorescence. The quantification of amyloid formation was further performed through ThT-induced fluorescence analysis. Sample preparation was performed using the same method as for CR binding. Each sample was placed in the wells of a 24-well plate that were filled with 0.5 mL of 50 µM ThT solution in 20 mM Tris-HCl (pH 8.0). The intensity reading was taken at 450 nm for λex and 486 nm for λem in triplicate using the Victor 3 microplate reader. Effects of Temperature during/after Fibrillation. To investigate the effect of temperature on fibrillation, PrP106-126 peptideimmobilized glass slides were incubated in a 100 µM solution at 37, 50, and 70 °C for 72 h. For the study of heat stability of preformed fibrils, samples incubated at 37 °C were further incubated in 200 mM acetate buffer (pH 5.5, 150 mM NaCl) at either 37 or 100 °C for 48 h. The morphology of each sample was analyzed by ex situ AFM.
Results and Discussion For the solid surface-based fibrillation of prion peptides, we immobilized the peptides on an NHS-activated glass slide, and the slide was further incubated in a solution that contained fresh prion peptide monomers. To compare the fibrillation of prion peptides on the solid surface with that in solution phase, the peptide solution was incubated for 72 h at 37 °C with or without the prion peptide-immobilized surface. According to our observation using ex situ AFM, the number density of prion fibrils formed in the solution phase was quite low and varied from sample to sample (Figure 1B). In contrast, when prion fibrils were formed on the solid surface, all of the analyzed spots contained numerous fibrils with similar shapes, indicating that the solid surface-based system is a highly efficient route for prion fibrillation in vitro. When we analyzed the prion peptide solution where the prion peptide-immobilized template was incubated, only small aggregates and a few short fibrils were observed, which confirms that fibrils found on the solid surface did not originate from the adsorption of preformed fibrils in the solution phase (Figure S1 in Supporting Information). The number of fibrillar structures increased with incubation time, and the whole surface of the analyzed area was covered with prion peptide fibrils after 72 h of incubation (Figure 2). According to the cross-sectional analysis of ex situ AFM images, the average diameter of fibrils was 5-8 nm with a length of about 250 nm. These numerical values are in good agreement with previous studies that showed the characteristics of
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Figure 3. (A) Time-dependent CD spectra and (B) secondary structure analysis of PrP106-126 fibrils. The template was incubated in peptide solution (800 µM) at 37 °C for 0, 24, 48, and 72 h.
Figure 4. Time-dependent changes in (A) absorbance at 535 nm by Congo red binding to the fibrils and (B) ThT-induced fluorescence of the fibrils. The template was incubated in PrP106-126 solution (200 µM) at 37 °C for 0, 24, 48, and 72 h. Data were analyzed by the Student’s t test (*, p < 0.001 when compared to the 0 h sample).
PrP106-126 fibrils with >100 nm length and 6-10 nm diameter.21,22 The effects of surface properties on prion peptide fibrillation were also investigated using different kinds of selfassembled monolayers (SAMs) (Figure S2). We immobilized prion peptides onto the surface with different SAMs, which include (3-aminopropyl)-triethoxysilane, dextran-500 kDa, polyL-lysine, 6-mercaptohexanoic acid, 11-mercaptoundecanoic acid, and 16-mercaptohexadecanoic acid. According to our observation, the formation of prion fibrils was more highly accelerated than in the solution phase for all kinds of tested SAMs with negligible differences in the morphology of fibrils; this result indicates that prion peptide fibrillation was not influenced by the surface type once prion peptides were seeded onto the surface prior to incubation. To confirm whether the fibrillar structures formed on the solid surface had amyloid-like properties, the secondary structure of fibrils was investigated using CD (Figure 3). Amyloid fibrils typically have dominant cross β-sheet structures,23 and the characteristic CD spectra of the β-sheet are known to consist of a negative minimum peak at 218 nm and a positive maximum peak at 195 nm.24 According to the CD analysis of prion fibrils self-assembled on the solid surface, the spectra exhibited typical features of β-sheet structure with increased incubation time (Figure 3A). The intensity of the maximum peak became positive (21) Florio, T.; Paludi, D.; Villa, V.; Principe, D. R.; Corsaro, A.; Millo, E.; Damonte, G.; D’Arrigo, C.; Russo, C.; Schettini, G.; Aceto, A. J. Neurochem. 2003, 85, 62–72. (22) Kuwata, K.; Matumono, T.; Cheng, H.; Nagayama, K.; James, T. L.; Roder, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14790–14795. (23) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128–8147. (24) Kelly, S. M.; Jess, T. J.; Price, N. C. Biochim. Biophys. Acta 2005, 1751, 119–139.
with a shift toward 195 nm, whereas the minimum peak shifted from 210 to 214 nm with its intensity being negative for all testing times. The content of β-sheet structure increased from 42.3 to 62.2%, whereas the percentage of random coil structure decreased from 57.7 to 35.8% with incubation (Figure 3B). For all cases, the content of R-helix or β-turn secondary structures was kept at 100 °C in an aqueous solution. Ure2p, a yeast prion protein, also partially aggregated after being heated to 100 °C as a result of the denaturation and recongealing of the C-terminal domain.29
Conclusions We succeeded in developing a solid-based in vitro system for prion amyloid fibrillation. Prion peptides self-assembled into amyloid fibrils on the solid surface, which was confirmed by multiple analyses using ex situ AFM, CD, and amyloid-specific dyes such as CR and ThT. We found that the self-assembly of prion peptides into amyloid fibrils was greatly accelerated on the solid surface regardless of the surface type, with resulting fibril (29) Baxa, U.; Ross, P. D.; Wickner, R. B.; Steven, A. C. J. Mol. Biol. 2004, 339, 259–264.
structures being more uniform than those formed by the conventional solution-based method. Our solid surface-based in vitro system can be applied to high-throughput analysis using microarray10 or microfluidics30 for screening environmental factors and chemicals affecting prion fibrillation. Acknowledgment. This research was supported by grants from the BioGreen21 Program (20070301034038) and the Korea Science and Engineering Foundation (KOSEF) NRL Program (R0A-2008-000-20041-0). Supporting Information Available: Representative ex situ AFM images of fibrils formed in the solution phase where the template was incubated, and on prion peptide-seeded SAM surfaces. This material is available free of charge via the Internet at http://pubs.acs.org. LA802931K (30) Lee, J. S.; Park, C. B. Langmuir 2008, 24, 7068–7071.
