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Copyright © 2006 American Chemical Society Peggy Rigou,* Human Rezaei, Jeanne Grosclaude, Siobhán Staunton, and Hervé Quiquampoix Virologie et Immunologie Moléculaires, INRA, F-78352 Jouy-en-Josas, France, and UMR Rhizosphère et Symbiose, INRA-ENSAM, 2 Place Pierre Viala, 34060 Montpellier Cedex 01, France Received for review August 26, 2005 Revised manuscript received January 6, 2006 Accepted January 6, 2006 Abstract: Prions, the infectious agents thought to be responsible for transmissible spongiform encephalopathies, may contaminate soils and have been reported to persist there for years. We have studied the adsorption and desorption of a model recombinant prion protein on montmorillonite and natural soil samples in order to elucidate mechanisms of prion retention in soils. Clay minerals, such as montmorillonite, are known to be strong adsorbents for organic molecules, including proteins. Montmorillonite was found to have a large and selective adsorption capacity for both the normal and the aggregated prion protein. Adsorption occurred mainly via the N-terminal domain of the protein. Incubation with standard buffers and detergents did not desorb the full length protein from montmorillonite, emphasizing the largely irreversible trapping of prion protein by this soil constituent. An original electroelution method was developed to extract prion protein from both montmorillonite and natural soil samples, allowing quantification when coupled with rapid prion detection tests. This easy-to-perform method produced concentrated prion protein extracts and allowed detection of protein at levels as low as 0.2 ppb in natural soils. Introduction Proteins are macromolecules with large affinities for water-solid interfaces, since they are flexible polymeric chains with lateral groups having contrasting physicochem ical properties: hydrophilic or hydrophobic, negatively, neutrally, or positively charged lateral chains. As a result, proteins have strong and complex interactions with the extensive mineral and organomineral surfaces in soil (12-16). The complexity of the various types of interactions involved makes the prediction of the interaction of a given protein with the different components of soil surfaces extremely difficult. For example, the -glucosidase of Aspergillus niger does not adsorb on the surface of montmorillonite above pH 6 (17), nor does bovine serum albumin above pH 6.5 (18). Acid phosphatases of ectomycorrhizal fungi have particularly diversified behavior in the presence of soil clays, from total adsorption, observed for Suillus mediterraneensis, to total repulsion, for Pisolithus tinctorius (19). The interaction of prion protein with soil fractions has been currently investigated, as reported at recent conferences (20). On other solid surfaces, the prion protein binds exceptionally strongly to plastic or metal surfaces encountered in medical and chirurgical devices (21-24), which has led to specific provisions for prion decontamination (25). The prion protein is thus expected to display a unique behavior on soil organic or mineral solid surfaces. The key event in the pathogenesis due to the prion is the conversion of the -helix-rich host prion protein (PrPC or PrPsens) into a pathogenic isoform (PrPSc or PrPres) characterized by its insolubility, its higher -sheet content, and its protease resistance (PrPres) (26). Adsorption of the prion protein onto solid components might explain trapping and the persistence of infectivity in soil. Detection of prion protein bound to soil mineral surfaces is a prerequisite to the monitoring and control of environmental contamination. Direct immunochemical detection of proteins adsorbed to mineral or organic material is impeded by the strong nonspecific adsorption of the antibodies to the matrix (27). No EPA-established standard method exists for the extraction and detection of the prion pathogen in soil (28). We believe that a better understanding of the adsorption and desorption of the prion protein on specified solid components such as clay is required in order to develop reliable methods for the extraction of trapped prion protein and the detection of contamination. We first investigated the interaction of a model prion protein (recombinant purified prion protein, recPrP) in a simple system, namely montmorillonite, a model clay mineral, revealing a unique and selective trapping capacity for prion protein. This adsorption was quasi-irreversible, and other proteins did not compete efficiently. We further developed an original technique of electroelution that allowed simultaneous extraction, concentration, and quantitative detection of recPrP in montmorillonite as well as natural soil samples. Experimental Procedures 2. Clay Mineral Montmorillonite. The clay mineral montmorillonite (Mt) is a common constituent of the mineral phase of soils with a very large adsorption capacity due to its large negatively charged surface area. In aqueous suspen sion the mineral occurs as submicron-sized particles. An aqueous colloidal suspension (20 g/L) of the clay-sized fraction (<2 m) of a Wyoming Mt saturated with sodium, with a specific area of 800 m2/g, was prepared and stored refrigerated. Aliquots of suspension were diluted and used for assays. 3. Natural Soil Samples. Soil is a complex matrix due to the presence of mineral, organic, and microbiological components that vary with soil type. Three soils were used in this experiment, a sandy topsoil sample, identified in previous work as Silwood soil (soil 1), obtained from the Department of Environmental Science and Technology at Imperial College, London (G. Shaw and C. Cooke), (35-37), and two topsoil samples, a sand and a sandy loam (soils 2 and 3), from the DEFRA (UK Department for Environment Food and Rural Affairs) field lysimeter experiment set up by the Neuropathogenesis Unit at the Institute of Animal Health, Edinburgh (R. Somerville). Soil particle size compositions were as follows: soil 1, 6% clay, 6.3% silt, 87.7% sand; soil 2, 6% clay, 8.3% silt, 85.7% sand; soil 3, 16% clay, 14% silt, 70% sand. All soils are currently being used in studies of prion transport. Samples were considered as initially free of prion contamination on the basis of (1) the place where they were initially collected (no pasture, no manure) and (2) the absence of signal when submitted to electroelution and prion protein detection. They were air-dried for 24 h, sieved to <2 mm, and stored at 4 C prior to use. 4. Spiking Method. Montmorillonite Samples. A constant mass of 15 g of -coiled or -sheeted recPrP or 30 g of truncated recPrP (C-terminal part) in sodium acetate buffer (20 mM at pH 5) were spiked onto various amounts of montmorillonite from the stock solution at 20 g/L and diluted to a final volume of 500 L of sodium acetate buffer. Natural Soil Samples. Aliquots of 0.25 g of dry soil were wetted with 100 L of deionized water and put into contact with 25 g of recPrP. Spiked soil was homogenized in a vortex and incubated for up to 2 months, when necessary, at constant temperature. 5. Adsorption/Desorption of recPrP with Montmoril lonite. After incubation for 2 h at room temperature, recPrP-spiked samples of montmorillonite suspension were centrifuged for 15 min at 14 000g. Montmorillonite particles were deposited as a pellet (P1) and were easily separated from the supernatant solution (S1). Supernatant solutions were retained for a back-titration of the nonadsorbed protein by Western blot analysis. Desorption experiments were performed on the Mt pellets (P1) by resuspension in 100 L of solution containing one of the desorbing reagents listed in Table 1 using a vortex. After a contact time of 10 min, samples were again centrifuged for 15 min at 14 000g, giving supernatants (S2) that were recovered and analyzed by Western blot for the detection of recPrP that may have been desorbed from the solid matrix. When using Laemmli buffer 4× (250 mM Tris pH 6.8, 5% SDS, 100 mM DTT) for desorption, samples were boiled for 2 min before centrifugation. 6. Competitive Adsorption. Competition for adsorption was tested by the simultaneous addition to clay (0.3 mg) of various amounts (from 6 g to 3 mg of total protein) of fetal calf serum (FCS), a mixture of macromolecular proteins, in the presence of 6 g of full-length prion protein. The ratio of recPrP/total FCS proteins thus varied from 1/1 to 1/500. The 1/50 ratio of recPrP/Mt was chosen since recPrP was completely adsorbed in the absence of FCS. After 1 h of contact at room temperature, samples were centrifuged for 15 min at 14 000g. The supernatant solutions were recovered for Western blot analysis of the remaining FCS and recPrP proteins in solution. 7. Western Blot Detection of recPrP. A Western blot method was used as a semiquantitative method of analysis. Aliquots of supernatants (7 L) were added to Laemmli buffer 4× (3 L), boiled, and loaded onto a sodium dodecyl sulfate (SDS) polyacrylamide gel (12% acrylamide) in a miniprotean II Bio-Rad electrophoresis system. The gel was transferred onto a Protron nitrocellulose membrane in a minigel transblot all system (Bio-Rad). The blots were revealed with 2D6, a monoclonal antibody (29) as a primary antibody and goat anti-mouse IgG immunoglobulins conjugated to peroxidase as secondary antibody (P.A.R.I.S, Compiègne, France). The 2D6 antibody is directed against the 146-182 region, in the C-terminal domain of the ovine protein. Quantities down to 10.5 ng of recPrP deposited on the gel could be detected by this method (Figure 1). -------------------------------------------------------------------------------- 8. Electroelution of recPrP Adsorbed on Montmorillonite and Soil Samples. The recPrP-spiked soil samples were boiled in 100 L of Laemmli buffer 4× and mixed with melted agarose 6 ” (agarose electrophoresis grade, Invitrogen) in Tris (25 mM)-glycine (20 mM) buffer pH 7.8 and then poured into cylindric glass tubes closed at the bottom extremity with a cellulose membrane (nominal molecular weight cutoff 6000-8000). After cooling, a 20% acrylamide gel disk was formed on top of the soil/agarose column, thus creating a Tris-glycine/0.1% SDS anodic reservoir (50-100 L) sealed with a cellulose membrane, in which eluted recPrP could be concentrated. Glass tubes were then cast horizontally in agarose (1% in water), forming a conductive block in an electrophoresis apparatus (Fisher Scientific). Overnight electroelution was then performed in Tris-glycine/SDS buffer under an electrical tension of 50-80 V. The protein eluted and concentrated in the reservoir was recovered for Western blot or immunochemical ELISA quantification. 9. ELISA Analysis. Quantitative analyses of electroeluted recPrP from montmorillonite and soil samples were performed using the Institut POURQUIER-Scrapie ELISA test (Montpellier, France) (38). Samples and peroxidase coupled monoclonal antibody (AcM) were directly incubated in Elisa plaques coated with one capture antibody. After washing of the plaques, a luminescent signal was obtained with the addition of luminol. The intensity of the signal was representative of the quantity of recPrP in the sample. The suppliers report the detection limit of the system to be 80 pg/mL for native PrP using recommended diluents. The calibration curve obtained for recPrP diluted in the electroelution recovery buffer (Tris-glycine/SDS) showed a limit of quantification down to 250 pg/mL of recPrP (Figure 2). -------------------------------------------------------------------------------- Results Back-titration on aliquots of supernatants S1 by Western blot analysis showed no signal of full-length recPrP when the adsorption was performed at recPrP/Mt ratios between 1/500 and 1/1 (w/w) (Figure 3a). Given the limit of detection of the Western blot method, the absence of recPrP signal indicated that there was less than 10.5 ng of full length recPrP in the 7 L aliquot loaded onto the gel; hence, not more than 750 ng remained in the whole supernatant solution, corre sponding to 5% of initially added protein. Thus, at least 95% of added recPrP adsorbed within 10 min onto the Mt in samples with recPrP/Mt ratios ranging from 1/500 up to 1/1 (w/w). When recPrP was present in large excess (recPrP/Mt ratio 2/1 w/w), adsorption onto Mt was not complete, since recPrP could be detected in the supernatant solutions (Figure 3a). The interaction of the oligomeric -sheet form with montmorillonite was studied in the same range of recPrP/Mt ratios. No -sheeted recPrP was detected in the supernatants S1 (Figure 3b) when the adsorption was performed at recPrP/Mt ratios between 1/500 and 1/1 (w/w). As observed for the full-length recPrP, the -recPrP was not fully adsorbed on Mt when added in large excess, with large quantities of recPrP being detected by Western blot (Figure 3b). -------------------------------------------------------------------------------- The full-length protein was not desorbed from Mt pellets P1 by simple addition of PBS (phosphate-buffered saline) or various detergents (see Experimental Procedures, section 5) (Figure 4). However, with the addition of Laemmli buffer 4×, degradation fragments (C-terminal) could be partially recovered and detected. -------------------------------------------------------------------------------- 2. Involvement of recPrP Domains in the Adsorption on Mt. To elucidate the cause of the exceptional affinity of the recPrP protein to Mt, we compared the adsorption properties of the full-length protein and the C-terminal fragment. At a protein/clay ratio of 1/250 (w/w), no signal of full-length recPrP nor C-terminal fragment was detected in supernatant solution by Western blot analysis (Figure 5), indicating complete adsorption of both the C-terminal fragment and the full length recPrP. However, when less Mt was present (recPrP/Mt ratio of 1/25), the C-terminal fragment was detected in the supernatant solution, while full-length recPrP was still not detected (Figure 5a). So the N-terminal part of the protein is an important determinant of the interaction with Mt. -------------------------------------------------------------------------------- 3. Competitive Adsorption of Proteins. The results presented above demonstrated that adsorption of prion proteins on montmorillonite was strong and largely irrevers ible. However, in soil, other proteins are continuously released by biological systems and form coatings on mineral surfaces. These may compete with the prion protein for adsorption. We therefore studied the simultaneous adsorption of prion protein and fetal calf serum (FCS), which contains a large mixture of proteins. We measured the adsorption of 6 g of full-length prion protein on montmorillonite in the presence of 6 g to 3 mg of FCS. No prion protein was detected by Western blot analysis of the supernatant solutions at any concentration of serum proteins, even when FCS was present in excess and could be detected in solution, indicating that the Mt surface was saturated (ratio FCS/recPrP of 500/1 w/w) (Figure 6). -------------------------------------------------------------------------------- 4. Electroelution of recPrP Adsorbed to Montmorillonite and Natural Soil Samples. The poor desorbability of recPrP protein from minerals is a considerable barrier to its detection in natural soil systems. Taking as our starting point the somewhat better extractability by Laemmli buffer and the negative charge conferred by this detergent to the full-length protein, we developed a simple robust technique combining extraction, transport of the negatively charged recPrP under an electrical field, concentration of the eluted protein in a small volume, and detection of recPrP by Western blot or commercially available ELISA kits. Figure 7a shows that 15-25% of adsorbed recPrP was reproducibly extracted from montmorillonite and detected by this method. A smaller fraction of the -sheeted recPrP was extracted (5%). For natural samples, 10-40% of spiked recPrP was extracted and detected from the three different soils investigated (Figure 7b). The electroelution method was more efficient than extraction in Laemmli buffer alone, as seen from the data presented in Figure 8 for the three soils spiked with a mixture of full-length recPrP and the C-terminal part. While the buffer alone extracted only the C-terminal part, when combined with electroelution, both proteins were quantitatively and reproducibly extracted. -------------------------------------------------------------------------------- Duplicate samples of montmorillonite spiked with recPrP were incubated for periods between 1 day and 2 months and then submitted to electroelution. The quantitative results of the ELISA analyses of the supernatant solutions are shown in Figure 7a. After the first week the fraction of initially added recPrP extracted decreased continuously to 1% after 2 months under nonsterile conditions. Similarly, the extractable fraction of the -sheeted recPrP was no longer detectable after 1 week. Duplicate samples of each of the three soils were also spiked with full-length recPrP, incubated for up to 2 months under nonsterile conditions, and then electroeluted. For each of the soils, extractability decreased with increasing incubation period. Discussion A previous study under laminar flow conditions has shown that recPrP was irreversibly adsorbed on muscovite mica surface (39). Montmorillonite and muscovite are both phyllosilicates sharing some common surface properties, such as a pH-independent electronegative basal surface charge and a siloxane external layer (18, 40). Thus, our results can be interpreted according to several consistent interpretations of the interaction of proteins with solid surfaces in general (41, 42) or soil mineral surfaces in particular (12, 13) based on the importance of both Coulombic and hydrophobic interactions. Protein affinity for solid surfaces can be explained by (i) an enthalpic component to the Gibbs energy decrease of the system when Coulombic interactions are attractive and, additionally (but less importantly), to the attractive Lifsh!tz-van der Waals interactions and (ii) an entropic component that can be decomposed under hydrophobic interactions with the surfaces and with changes of the conformational entropy of the protein (13, 41). The hydrophobic interactions of proteins with surfaces such as montmorillonite, a swelling clay, could seem paradoxal but are due to the siloxane surface. When hydrophilic exchangeable cations on the montmorillonite surface are exchanged by the positively charged amino acids side chains of proteins, the interface of interac tion is the hydrophobic siloxane layer (18, 40). The adsorption process is frequently accompanied by either pH-dependent orientation toward clay surfaces (43) or by pH-dependent changes in the macromolecular structures (44). The interaction of prion protein with montmorillonite is in agreement with these general mechanisms. Changes in secondary structures have been previously observed by FTIR spectroscopy for recPrP adsorbed on montmorillonite, showing a loss in -helices concomitant to an increase in -structures at any pH above 4 (45). In the present study, two observations allowed us to conclude that adsorption on montmorillonite took place largely via the positively charged N-terminal domain of recPrP. First, a greater amount of full-length recPrP was adsorbed than was the C-terminal domain fragment. Second, it was possible to desorb the C-terminal fragment with Laemmli buffer, while none of the buffers and detergents tested were able to extract the full-length recPrP (Figures 4 and 5). The electrostatic interaction between the protein and the negatively charged montmorillonite is most probably the origin of the strong adsorption of the full length recPrP. Accordingly the structural arrangement of the -sheeted oligomer, known to expose mostly the N-terminal moieties (34), should favor stronger interactions with the clay than those observed with the -recPrP. Indeed, stronger resistance to electroelution was observed for the oligomer. However, since saline buffers do not elute full-length recPrP nor C-terminal fragment from montmorillonite and since for the latter denaturing agents such as Laemmli buffer are required, other types of interactions contribute to this strong adsorption. This investigation of the prion protein adsorption to montmorillonite clay, an important constituent of mineral phase of soil, lends further support to the assumption that soil may be an intermediate in animal-to-animal transmission of TSE disease (1, 5, 6). In the environment, prion protein adsorbed to soil particles are unlikely to be desorbed and washed through the soil horizons or diluted to contaminate groundwater, unless colloid-facilitated transport is involved. The presence of other proteins naturally found in the environment should not compete with or reverse prion protein adsorption to soil components. Prion strongly complexed with solid colloidal particles at the soil surface is very likely to be reingested by animals through grazing or drinking in puddles. Progressive accumulation of trace amounts of prion on these high-trapping particles might constitute localized "infectivity hot points" in the environ ment, explaining previous field observations (1, 5, 6). Adsorbed infectivity on steel wire is transmissible to intact brains or cells in culture (22, 23). Whether or not clay-adsorbed infectivity is actually transmissible to animals is currently being investigated on transgenic mice. Nevertheless, our observations demonstrate that clay minerals are efficient trapping systems for prion proteins, allowing the selective concentration and removal of pathogenic prions from biological fluids. Soil microorganisms play a major role in the degradation of organic matter, and some environmental bacteria have been reported to cleave protease resistant prion protein (46). The antigenic degradation of adsorbed prion protein by soil microorganisms may explain, at least in part, our observation that apparent recPrP recoveries from nonsterile natural soil samples decreased with increasing protein-mineral contact period. The recPrP may also become progressively trapped in less accessible interstitial sites, such as clay interlayer spaces and other soil micropores (47). Prion dissemination in the environment will result from the balance between solid surface adsorption and biotic factors, which are prone to generate soluble protein fragments. Advances on physical mechanisms and biological activity in soils should open the way to an integrated view about the fate of prion in soil and finally lead to the risk assessment of prion persistence in the environment. Methods for the control and assessment of prion contamination at sites potentially at risk (whelping areas for example) are urgently needed. The original electroelution technique presented in this work allows sensitive prion detection in natural soils. The method was based on the combined effect of the denaturing detergent Laemmli buffer that conferred a negative electric charge to the protein, together with a dynamic transport system under an electrical field. It has several advantages: (i) it is simple to set up and perform, (ii) it is an environmentally friendly method that does not require the use of toxic materials nor generate toxic waste, (iii) it yields concentrated extracts of recPrP material, and (iv) it allows the detection of quite low levels of extracted protein. Considering the limit of detection of ELISA method down to 250 pg/mL in the elution buffer, an average extraction recovery of 25% and that recPrP after extraction can be concentrated down to a volume of 50-100 L, the electroelution method coupled with immunochemical detection kits offers a detection limit down to 12.5-25 pg of protein extracted from 250 mg of soil sample (0.2 ppb). Recoveries vary with the type of soil, and more work is required to determine which soil properties, apart from texture and mineral composition, determine extractability. This suc cessful electroelution process, applicable to soils of contrast ing texture and to pure clay, overcomes the difficulties encountered with detergent-based methods. This system of extraction could be scaled up and adapted for the extraction of larger soil samples. In the case where a soil might be suspected to have been contaminated with infected material or simply with specified risk material, this technique would allow the mapping of the contaminated area. This electroelution technique could be adapted for the extraction of recPrP from different matrixes or materials (for example, the decontamination of medical or chirurgical devices) and is also suitable for the extraction of other undesirable biological compounds present in the environment. 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