1 Federal Research Centre for Nutrition, Haid-und-Neustr. 9, 76131 Karlsruhe, Germany
2 Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, 72076 Tübingen, Germany
Correspondence
Avelina Fernández García
veli.fernandez{at}bfe.uni-karlsruhe.de
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ABSTRACT |
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MAIN TEXT |
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These experiments were performed with a single brain pool of the 263K strain of hamster-adapted scrapie agent. Brains containing PrPSc were homogenized in a 1 : 10 dilution of PBS, pH 7·4, in a FastPrep cell disrupter (Qbiogene). Sets of duplicate samples were heated at 60 °C and/or pressurized up to 1000 MPa for 2 h independently. Total volumes of 250 µl of homogenate (10 %, w/v) were pressurized in a hydraulic press, U 101 (Polish Academy of Sciences, Warsaw). U 101 is a manually operated twin-piston hydraulic press (100 mm piston length, 80 mm piston movement). The vessel is a cylinder made of steel, with an inside diameter of 16 mm and 150 mm in height. The piston position is monitored with a linear transformer transducer and the pressure-measuring unit is an in-vessel manganin pressure gauge; both are digitally displayed. The pressure-transmitting medium was a 7 : 3 mixture of petroleum ether (boiling point 80100 °C) and hydraulic oil (SAE 32). Samples were pressurized in polyethylene caps hermetically closed by heat sealing. The effects of adiabatic heating were minimized by the long pressure rise necessary to achieve the highest pressures (at least 150 s above 700 MPa) and by continuous control of the temperature in the vessel using a thermostat (Polystat) coupled to the cylinder. The effects of pressure are discussed in relation to the pressure intensity, since the holding time was always 2 h.
Samples (15 µl) were subsequently digested with PK at 73 µg ml-1 final concentration (Sigma) for 1 h at 37 °C. Triplicates of each sample were examined. Positive controls were samples not treated with PK. After denaturation for 5 min at 95 °C, the samples were separated by electrophoresis through 10 % polyacrylamide gels. Separated proteins were finally electrotransferred to PVDF membranes (0·2 µm pore size; Bio-Rad). Surplus binding sites were blocked by incubating the membranes in 5 % non-fat dry milk and 5 % BSA in PBS with 0·1 % Triton X-100. Membranes were then incubated with the anti-hamster PrPSc 3F4 monoclonal antibody (Signet Laboratories) diluted 1 : 5000 in blocking solution. After incubation with antibodies, membranes were washed extensively with PBS/0·1 % Triton X-100 and incubated with peroxidase-labelled goat anti-mouse IgG (Oncogene) diluted 1 : 3000 in PBS/0·1 % Triton X-100. After further washing, antibody binding was visualized on a highly sensitive Hyperfilm ECL (Amersham) using the enhanced chemiluminescence detection system.
Infectivity bioassays were performed with the temperature-/high-pressure-treated materials following an incubation time interval protocol (Prusiner et al., 1982). After pressurization, 20 µl hamster brain homogenates (10 %, w/v) were intracerebrally inoculated into groups of four or five weanling hamsters. Untreated or heated samples (2 h at 60 °C) of the same brain homogenate were examined as controls. Hamsters were observed for clinical signs of scrapie for a period of 180 days and killed immediately after showing disease symptoms. Brains from killed hamsters were removed and frozen at -70 °C. Subsequently, they were assayed to detect the PK-resistant PrPSc.
The various treated and untreated brain homogenates were incubated with (+) or without (-) PK and analysed in Western blots. As shown in Fig. 1, the non-pressure-treated control at 20 °C showed the typical pattern representing the various glycoforms of the
-aggregated prion protein resistant to digestion with PK (2032 kDa) and the typical band shift after PK treatment. The same characteristic glycoform pattern was also detected in homogenates treated at 60 °C (Fig. 1
) showing the same resistance to digestion with PK. As expected, temperatures lower than the recommended sterilization conditions had no influence on the PK resistance of the infectious agent.
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In contrast, no PrP-specific proteins could be identified after PK treatment following pressure conditions over 500 MPa, indicating that the hamster prion protein was efficiently digested with PK (Fig. 1). These results may be explained by the shape of typical pressure-stability contours of proteins. They show that at certain pressure/temperature levels (usually over 500 MPa), proteins undergo changes in their tertiary structure, affecting functionality. If the pressure is high enough, proteins denature (Mozhaev et al., 1996
).
If only samples containing PK-resistant prions are considered pathological, the samples pressurized at or above 500 MPa should have lost potential infectivity. To test this, infectivity bioassays were performed in hamsters by intracerebral inoculation of the treated homogenates. Typically, signs of scrapie become prominent 7090 days after hamster inoculation. Animals in this experiment were observed for up to 180 days. Data obtained are presented in Table 1. Untreated crude brain hamster homogenates and those treated at 60 °C and/or 100/200 MPa led to terminal TSE disease in all of the hamsters after 7990 days. There were no significant differences in infection among these hamsters, suggesting a negligible inactivation rate under these conditions.
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There are several publications on the effects of pressure (from 50 to 1200 MPa) on the protein folding of several amyloid-forming proteins, such as lysozyme (Akasaka et al., 1997), transthyretin (Ferrao-Gonzales et al., 2000
) and amyloid A (Dubois et al., 1999
), but little is known about pressure-induced prion unfolding. Kuwata et al. (2002)
identified a metastable transitional conformer of PrPC using two-dimensional nuclear magnetic resonance measurements under variable pressure (up to 250 MPa). Additionally, the relative pressure sensitivity of the cellular form of the hamster prion protein has been reported by Torrent et al. (2003)
, but the possible infectivity of the pressure-induced intermediate states was not assayed. Pressure has also been reported to induce the aggregation of transthyretin, converting the native protein into a tetrameric, amyloidogenic state. However, Zhou et al. (2001)
found that only the combination of pressure with a denaturing agent (guanidinium hydrochloride) was able to induce an irreversible unfolding in the structure of the yeast prion protein Ure2, a similar phenomenon to that observed in the case of lysozyme. Pressure alone was inefficient, at least up to 600 MPa. Although the His-tagged Ure2 used for these experiments had amyloid-forming ability in vitro, the results disagreed with the irreversible effects on PrPSc structure shown in this work.
We are aware of only one paper dealing with the effect of pressure on infectious prions. Brown et al. (2003) reported a method based on the ultra-high pressure-assisted thermal sterilization of infectious hamster proteins. Within the proposed technique, initial temperatures were shifted up to 135142 °C due to adiabatic heating: ten 1 min cycles were as effective as 5 min of steam sterilization, an effect that could not be attributed to pressure. The irreversible effects related here are most likely due to changes in weak inter- or intramolecular interactions, since pressure is not able to break covalent bonds. These would first affect the stability of the cross-
structure of amyloids, increasing sensitivity to digestion with PK. Similar effects have been reported by Riesner et al. (1996)
, where SDS caused the disruption of prion rods, generating particles containing
-helices lacking infectivity. However, the experimental data mostly sustain the idea of an extraordinary pressure resistance of secondary structures based on
-sheets. For instance, only very high pressures (1200 MPa) caused the dissociation of aggregated amyloid A into
-helical and unordered structures and a decrease in
-sheet content; this process was nevertheless reversible after decompression (Dubois et al., 1999
). If, as assumed, the resistance of PrPSc to proteolytic digestion was based on the
-structure and the quaternary disposition, the remarkable efficiency of pressures above 500 MPa at only 60 °C in increasing the sensitivity of the hamster prion protein to PK is difficult to explain following the existing views about the limited potential of pressure to induce conformational changes in
-sheets. Thus, our results argue for the hypothesis of a supplementary factor (postulated by Telling et al., 1995
) present in the tissue modulating prion propagation and stability: the hypothesized macromolecule, or its binding site to prion proteins, might be highly pressure sensitive.
High hydrostatic pressure appears to be a promising treatment for producing safe products at temperatures below the sterilization parameters, e.g. baby food, by gentle inactivation of potentially present PrPSc-positive materials. Furthermore, we expect this technology to help understand certain principles of protein misfolding, in particular the behaviour of amyloidogenic fibrils and the development of amyloidoses: the results presented here on the effects of relatively low pressures combined with mild temperatures on the structure of PrPSc-infected brain materials and the decreased infectivity of pressurised materials in vivo are encouraging. Further results obtained with bioassays using extensive doseresponse curves will be addressed in forthcoming papers, as well as other aspects, to determine the effects of the pressure rise/pressure release or the pressurization kinetics on the sensitivity of PrPSc materials.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, P., Rau, E. H., Johnson, B. K., Bacote, A. E., Gibbs, C. J., Jr & Gajdusek, C. (2000). New studies on the heat resistance of hamster adapted scrapie agent: threshold survival after ashing at 600 °C suggests an inorganic template of replication. Proc Natl Acad Sci U S A 97, 34183421.
Brown, P., Meyer, R., Cardone, F. & Pocchiari, M. (2003). Ultra-high-pressure inactivation of prion infectivity in processed meat: a practical method to prevent human infection. Proc Natl Acad Sci U S A 100, 60936097.
Dubois, J., Ismail, A. A., Chan, S. L. & Ali-Khan, Z. (1999). Fourier transform infrared spectroscopic investigation of temperature- and pressure-induced disaggregation of amyloid A. Scand J Immunol 49, 376380.[CrossRef][Medline]
Ferrao-Gonzales, A. D., Souto, S. O., Silva, J. L. & Foguel, D. (2000). The pre-aggregated state of an amyloidogenic protein: hydrostatic pressure converts native transthyretin into the amyloidogenic state. Proc Natl Acad Sci U S A 97, 64456450.
Heremans, K. & Smeller, L. (1998). Protein structure and dynamics at high pressure. Biochim Biophys Acta 1386, 353370.[Medline]
Kuwata, K., Hua, L., Yamada, H., Legname, G., Prusiner, S., Akasaka, A. & James, T. L. (2002). Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc? Biochemistry 41, 1227712283.[CrossRef][Medline]
Mozhaev, V. V., Heremans, K., Frank, J., Masson, P. & Balny, C. (1996). High pressure effects on protein structure and function. Proteins 24, 8191.[CrossRef][Medline]
Prusiner, S. B. (1991). Molecular biology of prion diseases. Science 252, 15151522.[Medline]
Prusiner, S. B., Cochran, S. P., Groth, D. F., Downey, D. E., Bowman, K. A. & Martinez, H. M. (1982). Measurement of the scrapie agent using an incubation time interval assay. Ann Neurol 11, 353358.[Medline]
Randolph, T. W., Seefeldt, M. & Carpenter, J. F. (2002). High hydrostatic pressure as a tool to study protein aggregation and amyloidosis. Biochim Biophys Acta 1495, 224234.
Riesner, D., Kellings, K., Post, K., Wille, H., Serban, H., Groth, D., Baldwin, M. A. & Prusiner, S. B. (1996). Disruption of prion rods generates 10-nm spherical particles having high -helical content and lacking scrapie infectivity. J Virol 1996, 17141722.
Telling, G. C., Scott, J., Mastrianni, R., Gabizon, R., Torchia, M., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. (1995). Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83, 7990.[Medline]
Torrent, J., Alvarez Martinez, M. T., Heitz, F., Liautard, J.-P., Balny, C. & Lange, R. (2003). Alternative prion structural changes revealed by high pressure. Biochemistry 42, 13181325.[CrossRef][Medline]
Zhou, J.-M., Zhu, L., Balny, C. & Perrett, S. (2001). Pressure denaturation of the yeast prion protein Ure2. Biochem Biophys Res Commun 287, 147152.[CrossRef][Medline]
Received 10 June 2003;
accepted 8 October 2003.