- Email: [email protected]
Organic polyanions act as complexants of prion protein in soil
Available online at www.sciencedirect.com
Biochemical and Biophysical Research Communications 367 (2008) 323–329 www.elsevier.com/locate/ybbrc
Organic polyanions act as complexants of prion protein in soil Maurizio Polano a,b, Claudio Anselmi a,c,*, Liviana Leita d, Alessandro Negro e, Maria De Nobili b a SISSA, Via Beirut 2-4, I-34014 Trieste, Italy Department of Agriculture and Environmental Sciences, Universita` di Udine, Via delle Scienze 208, I-33100, Italy c CNR-INFM-DEMOCRITOS, Via Beirut 2-4, I-34014 Trieste, Italy C.R.A., Centro di Ricerca per lo Studio delle Relazioni Pianta-Suolo, Sede Distaccata di Gorizia, Via Trieste 23, I-34170 Gorizia, Italy e CRIBI Biotechnology Centre, Universita` di Padova, Via U. Bassi 58/b, I-35131 Padova, Italy b
d
Received 13 December 2007 Available online 2 January 2008
Abstract The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity. 2007 Elsevier Inc. All rights reserved. Keywords: Chronic wasting disease; Humic acid; Infectivity of soil; Pollution of soil; Clay; Docking
Infectious proteinaceous particles causing transmissible spongiform encephalopathies (TSEs) have recently become matter of considerable environmental pollution and concern. TSEs are a class of fatal neurodegenerative disorders that affect animals and human beings, which include Creutzfeldt-Jakob disease in human beings, bovine spongiform encephalopathy in cattle, scrapie in sheep, and chronic wasting disease (CWD) in cervids. The ethiologic agent of TSEs is the prion, a proteinaceous particle composed mainly of PrPSc, the b-sheet-rich conformer of the cellular form of the prion protein (PrPC), a normal constituent of
Abbreviations: PrP, prion protein; MoPrP, recombinant murine prion protein; TSE, transmissible spongiform encephalopathy; CWD, chronic wasting disease; HS, humic substance; HA, humic acid; Mnt, montmorillonite. * Corresponding author. Address: SISSA, Via Beirut 2-4, I-34014 Trieste, Italy. Fax: +39 040 3787528. E-mail address: [email protected] (C. Anselmi). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.143
mammalian cells. Accumulation of PrPSc in the central nervous system of animals and humans leads to the manifestation of the disease [1]. PrPC is composed of about 250 amino acids, mainly in a-helical conformation and upon conversion to PrPSc, it is sufficient for the spontaneous formation of prions in mammals without the intervention of any exogenous agent [1]. This has been recently demonstrated with the production in vitro of the synthetic prion [2]. To date the diffusion of CWD in the natural environment is a matter of growing alarm in the USA. CWD was identified in 1977 as a TSE or prion disease, which appears to be freely transmitted among cervids by direct or indirect horizontal contact with infected biological material [3,4]. It seems possible, however, that under natural conditions prions can contaminate the environment through the soil. The scrapie infectious agent indeed persists in soil for years retaining its capability to activate misfolding of the native protein and infectivity [5]. Soil has been shown to retain the pathogenic agent for years in a
324
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
form that is experimentally transmissible to laboratory animals [6,7] and different soils have been shown to strongly retain prions [8]. The extent to which the prion is transmitted from a contaminated environment is unknown. Several articles have started to address the problem of prions in the soil [5,9– 11], but the present approaches show several drawbacks and technical difficulties, as soil is a complex, multi-component system of interacting materials, both mineral and organic. Most studies have so far been concerned with the adsorption capacity of clay minerals [9–11]. The protein appears to bind to the mineral surface with the permanent negative charges via electrostatic interactions and to undergo a pH independent conformational change in the adsorbed state, which leads to an increased b-sheet structure. No quantitative data on the adsorption capacity of the clay or on the effect of different cations on adsorption isotherms is available to date. In contrast, the contribution of soil organic components in adsorption has been comparatively neglected, as they are only a minor fraction on a weight basis. However, among organic molecules, humic substances (HSs) are natural polyanions that result among the most reactive compounds in the soil and possess the largest specific surface area [12]. Notably, the majority of mineral surfaces in soils and sediments are normally coated with organic matter [13] and the positively charged mineral surfaces are changed into negatively charged or their overall negative charge is enhanced [14]. Adsorption of HSs can modify the physicochemical properties of the underlying mineral surface, thus rendering hydrophilic mineral surfaces more hydrophobic and more capable of adsorbing organic contaminants [15]. In this work, we have studied the effect of HSs and ions on the adsorption of recombinant PrP onto different clay minerals. In fact, it is commonly admitted that recombinant PrP could serve as a model for the normal cellular prion protein PrPC [2]. Both experiments and theoretical modeling indicate that HSs participate in the adsorption of the PrP in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks with accessible polar residues. We show that HSs enhance the complexant capacity of soil, even if it is not clear if this raises or lower the prion infectivity. In contrast, our results suggest that studies that only focus on clays limit our knowledge about TSEs in the environment, because they completely neglect one of the most reactive components of soil and are therefore not representative of the actual situation.
Materials and methods Cloning of MoPrP. Full-length recombinant murine prion protein from 23 to 231 (MoPrP) was prepared in Escherichia coli according to a previously described strategy [16]. Concentration of MoPrP stock solutions was determined by UV absorbance spectroscopy, using a Varian (Carry 50) spectrophotometer and 1-cm path length quartz cuvettes. An extinction coefficient for MoPrP
of 2.7 mg 1 cm2 at 280 nm was calculated with the ProtParam tool on expasy (http://www.expasy.org). Preparation of clay suspensions. Clay minerals (Wyoming Na-montmorillonite (Na-Mnt) (SWy-1), Arizona Ca-montmorillonite (Ca-Mnt) (SAz-1) and kaolinite with a cation exchange capacity of 120, 76.4, and 9 c+ mol/kg, respectively) were suspended at a rate of 0.05 (Na-Mnt), 0.25 (Ca-Mnt) or 0.4 mg/mL (kaolinite) in either mQ water or PBS at different ionic strengths (1· and 0.1·). For kaolinite suspension, particles greater than 2 mm were allowed to settle out by gravity and discarded. Preparation of clay–HA complexes. 0.0122 g of HA (International Humic Substances Society (IHSS) peat reference IR103H-2) and 1 g kaolinite were suspended in 10 mL of solutions 1 mM NaOH and 2.5 mM CaCl2, respectively, and then mixed for about 2 h at room temperature. Suspension was then centrifuged at 4000 rpm for 5 min and dried at 40 C for 20 h. The dry material was ground to a fine powder and then the suspension 0.1405 mg/mL was prepared with mQ reagent grade water. Adsorption isotherms. Varying volumes of concentrated MoPrP stock solutions (about 35–37 lg/mL) were spiked into Eppendorf centrifuge tubes containing a fixed volume (0.2–0.5 mL) of mineral suspensions and a complementary volume of either mQ water or PBS to give a constant final volume of 2 mL at fixed solution chemistry conditions. Adsorption isotherms were measured in triplicate batch experiments. After 10 min shaking, the MoPrP/clay suspension was centrifuged for 95 min at 14,000g on an Eppendorf 5402 centrifuge. The amount of adsorbed protein was calculated by difference from the equilibrium concentration in solution (depletion method) [17]. Difference spectra were record using a Cary 50 spectrophotometer. Amounts adsorbed vs equilibrium concentrations were fitted to both the linearized form of the Freundlich equation and the Hanes–Woolf linearized form of the Langmuir equation [18]. Molecular docking. Taking into account the average molecular weight, charge density and the average number of functional groups of the standard HA adopted in the experiments, we designed a 3-D model molecule by randomly connecting the more common HA organic molecular fragments (Supplementary material). The automated docking procedure vs PrPC (PDB entry 1XYX) [19] was performed using the program AutoDock 3.0.5 [20]. One thousand different complexes were analyzed.
Results Equilibrium adsorption of MoPrP on clays Kaolinite and montmorillonite (Mnt) were chosen as model clays. Kaolinite is one of the most abundant minerals in soils and sediments. Mnt forms by crystallization from solutions high in soluble silica and magnesium and has a 2:1 layer structure in which Al3+ is the normal ion in the central octahedral sheet. However about one-eight of the octahedra contain Mg2+ and the surplus negative charge is neutralized by exchangeable cations adsorbed to the surface. Cohesion between sheets is not very strong and depends on the amount of water present. When wet conditions occur, water causes the clay to swell dramatically and internal negatively charged surfaces become accessible [21]. Fig. 1A shows the adsorption isotherms of MoPrP on different clay minerals. Different affinities for MoPrP are: Na-Mnt > CaMnt > kaolinite > quartz sand. The dependence of the binding capacity of the clay minerals can be explained on the basis of the different mineralogy of the clays, in particular it indicates the importance of both particle size and charge. Whereas kaolinite is made of relatively large non-
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
325
Fig. 1. (A) Adsorption isotherm of clays at pH 7.0 in water. X/M is the adsorbed amount of prion and Ce is the prion concentration in solution at equilibrium. (B) Adsorption isotherm of Na-Mnt in different buffer solutions. (C) Adsorption isotherm of MoPrP on pure kaolinite and on HA–kaolinite complex. (D) Adsorption isotherm of HA at pH 6.6 at different ionic strengths.
expanding crystals and therefore possesses a low number of charged absorption sites (as confirmed by Fig. 1A), Mnt consists of much smaller crystals, characterized by huge specific surface area (800 m3/g) and much higher negative charge density. The separation between individual platelets varies depending mainly on the type of interlayer cations present and the ionic strength of the surrounding solution. Na-Mnt and Ca-Mnt are expected to behave differently as saturation with a divalent cation does not allow the complete swelling of the clay. Fig. 1A shows how the adsorption capacity of Na-Mnt is higher than Ca-Mnt, according to the higher interlayer distance of the first with respect to the second. This enlightens the capacity of Na-Mnt of adsorbing the recombinant prion protein not only at the surface, but also inside the interlamellar space of the clay structure. In contrast, the adsorption on Ca-Mnt is likely to occur only on the external surface of the clay. Adsorbed MoPrP could not be removed from the clay, even by strongly denaturing solutions such as 1% SDS. Solution ionic strength can have an effect on the adsorption process because the interlayer is hydrated and cations within the interlayer may exchange with those in the external solution. In order to verify the dependence of the clay adsorption on the salt, we performed experiments using
either PBS buffer or a solution of NaCl with the same ionic strength. Fig. 1B shows that adsorption affinity of Na-Mnt vs MoPrP is lower when the clay is suspended in PBS with respect to water and to NaCl. Maximum adsorption capacity of the clay was observed in the NaCl solution (Fig. 1B). As PBS buffer contains NaCl, KCl, and Na2HPO4, it seems that both the affinity and the maximum amount of adsorption depend on the type of ions more than the actual ionic strength. In this case, it is likely that Na+/K+ exchange lowers clay expandability and makes internal surfaces less accessible. Effect of HSs Unfortunately, the exact composition of HSs is unknown, as they are complex mixtures of polyphenol–polycarboxilic acids with colloidal and supramolecular characteristics, which have never been separated into pure components [22]. Nevertheless, standard humic acids (HAs) can now be used to avoid the uncertainties due to their polydisperse nature and different extraction procedures. Fig. 1C shows that the kaolinite–HA complex greatly increases both the adsorption affinity and the maximum amount of absorbed protein with respect to pure kaolinite,
326
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
which weakly adsorbs prions (Fig. 1A). Notably, values for kaolinite–HA complex are in the same order of those of NaMnt, which is among the clays with the highest adsorption capacity (Fig. 1A and B). Also solid HA alone shows an enormous affinity for the prion protein at different ionic strengths (Fig. 1D), approximately twenty times that of Na-Mnt. Molecular interactions with HA in solution HSs cannot only be present in soil and sediments as solids, but are also dissolved in the soil solution or in surface waters. We examined the possibility of interactions of MoPrP with dissolved HA. The intense absorption band at 193 nm in the UV spectrum of MoPrP decreases linearly upon addition of HA (Fig. 2A and B) suggesting the occurrence of an interaction with the backbone of exposed amino acids. Complete titration is achieved at a HA/MoPrP ratio of about 4.8:1 (W/W) (Fig. 2A). It is also possible
that at larger HA/MoPrP ratios, humic molecules completely shield the protein preventing adsorption by chromophores. The absorbance at 228 nm increases upon addition of HA, reaching a maximum at an apparent molar ratio of about 1 (W/W) and declining thereafter for larger HA/ MoPrP ratios (Fig. 2B). The shoulder at 228 nm is generally attributed to the absorbance of Asp, Glu, Asn, Gln, Arg, and His side chains and to a weak transition of electrons in peptide bonds (210–230 nm). In the truncated form of MoPrP, exposed side chains of this types (>40% exposure) belong to the a1 helix (C class amino acids) and to the poorly structured region near the C-terminus (A class amino acids) [23]. Fig. 2B would be coherent with an increased solvent exposure of this type of side chains and in their subsequent shielding by humic molecules. The fact that the complete annealing of the adsorption at both 190 and 228 nm is reached for relatively close values of HA/ MoPrP supports a general shielding mechanism more than
Fig. 2. (A) Variation of the absorbance of MoPrP vs the concentration of HA. (B) Variation of the absorption at 228 nm of MoPrP at different concentration ratio with HA (W/W).
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
327
Fig. 3. Occurrence of PrPC amino acids at the interface with HA. Most relevant amino acids are labelled.
the binding to specific sites, whereas site specific interaction can occur at the beginning of the titration. Molecular docking In silico docking experiments were performed to characterize the nature of the interactions between HAs and
PrPC. HA can assume many different conformations and seem to bind PrPC without any apparent specificity. We therefore analyzed the docked complexes in order to find the amino acids that are more frequently at the interface with HA (Fig. 3), as it is supposed they could be more important for association. Due to the lack of binding specificity, practically all amino acids are interacting with HA
Fig. 4. (A) PrPC amino acids involved in the binding of HA. (B) Examples of PrPC–HA complexes; different HA conformations are colored in blue, red, yellow, orange, and green. (C) Representation of the electrostatic potential at the PrPC surface. Blue and red represents positive and negative values, respectively.
328
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
in some complexes. In order to discriminate among true positives and the background noise, we evaluated frequency statistical dispersion and considered only those amino acids of which the number of counts differs from the mean value more than one standard deviation. They mostly correspond to two regions of PrPC, situated within helices a1 and a2, respectively (Figs. 3 and 4A and B), which correspond to zones that have been suggested to be sensible in the process of conversion of PrPC toward the pathogenic form [24]. Notably, also UV absorption spectra suggest the occurrence of conformational changes of residues within helix a1 upon interactions with HA. Analysis of PrPC surface shows that electrostatics plays a major role in stabilizing PrPC–HA complexes, as interface regions corresponds to two maxima of the electrostatic potential (Fig. 4C). However, also polar residues play a major role in binding. In particular, Asn side chains are both donors and acceptors of hydrogen bonds and N171, N173, N174, and N181 at helix a2 are among the amino acids that more frequently bind HA (Fig. 3). Discussion Several studies have demonstrated prions can be retained in soil in a form that is able to cause the insurgence of TSEs even several years after the soil contamination. However, within the large spread of potentially infected lands, prion diseases have become endemic only in geographically limited regions [25]. Reasons for this remain unknown, but it suggests a role of the different kinds of soil in either enhancing or containing prion infectivity. In fact, as the latter depends on a conformational change of the prion itself, interactions with substances able to enhance the change could favor infectivity. In this context, a good knowledge of the mechanisms of retention of prion in soils is of great importance for controlling the possibilities of dissemination of TSEs. Several aspects play a role in this process, such as the efficiency of sorption and desorption of the prion in soil and its capacity to undergo conformational changes and/or degradation. Our results suggest that MoPrP adsorption in soil is driven both by HAs and clays. The nature of the clay seems to be important as well as that of the ions in solution, as it is likely they influence clay expandability and internal surface accessibility to the protein. This suggests that different types of soil with different clays and ions could involve different fates for the prion. However, HAs not only show the strongest affinity for MoPrP, but they also considerably enhance the adsorption capacity of clays. Our results provide evidence of direct interactions between HAs and prions and suggest HAs promote conformational changes in some prion residues, which are located in regions of relevance for the conversion to the pathogenic form. There is further evidence in the literature that polyanionic molecules induce an efficient conversion of PrPC in vitro, eventually enhancing fibril growth. In contrast, recent studies
seem to exclude the possibility of a a–b conversion from a non-pathogenic form of prion to a pathogenic form induced by the interaction with soil clays [9]. On the other hand, low contents of soil organic matter is often associated to a number of CWD cases among free-ranging cervids in USA [25], whereas other studies suggest that factors, as metal ions, which are known to sequestrate HSs in soils, promote the insurgence of CWD [26]. Of course, biological tests on animals are needed to elucidate whether interaction with HSs can lead to either protection or deactivation of prion infectivity in soil. Acknowledgments Authors thank Prof. Giuseppe Legname, Prof. Paolo Carloni (SISSA), and Prof. Catia Sorgato (University of Padua) for valuable advices. This work was carried out with a grant of the Ministry of Agricultural Policies, finalized project RIFAFERT. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 12.143. References [1] S.B. Prusiner, Early evidence that a protease-resistant protein is an active component of the infectious prion, Cell 116 (2004) S109–S111. [2] G. Legname, I.V. Baskakov, H.O. Nguyen, D. Riesner, F.E. Cohen, S.J. DeArmond, S.B. Prusiner, Synthetic mammalian prions, Science 305 (2004) 673–676. [3] E.S. Williams, S. Young, Spongiform encephalopathies in Cervidae, Rev. Sci. Tech. 11 (1992) 551–567. [4] E.S. Williams, M.W. Miller, Chronic wasting disease in deer and elk in North America, Rev. Sci. Tech. 21 (2002) 305–316. [5] B. Seidel, A. Thomzig, A. Buschmann, M.H. Groschup, R. Peters, M. Beekes, K. Terytze, Scrapie agent (strain 263K) can transmit disease via the oral route after persistence in soil over years, PLoS ONE 2 (2007) e435. [6] P. Brown, D.C. Gajdusek, Survival of scrapie virus after 3 years’ interment, Lancet 337 (1991) 269–270. [7] D.M. Taylor, Resistance of transmissible spongiform encephalopathy agents to decontamination, Contrib. Microbiol. 11 (2004) 136–145. [8] R.C. Angers, S.R. Browning, T.S. Seward, C.J. Sigurdson, M.W. Miller, E.A. Hoover, G.C. Telling, Prions in skeletal muscles of deer with chronic wasting disease, Science 311 (2006) 1117. [9] M. Revault, H. Quiquampoix, M.H. Baron, S. Noinville, Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays, Biochim. Biophys. Acta 1724 (2005) 367–374. [10] C.J. Johnson, K.E. Phillips, P.T. Schramm, D. McKenzie, J.M. Aiken, J.A. Pedersen, Prions adhere to soil minerals and remain infectious, PLoS Pathog. 2 (2006) e32. [11] L. Leita, F. Fornasier, M. De Nobili, A. Bertoli, S. Genovesi, P. Sequi, Interactions of prion proteins with soil, Soil Biol. Biochem. 8 (2006) 1638–1644. [12] N. Senesi, Binding mechanisms of pesticides to soil humic substances, Sci. Total Environ. 123–124 (1992) 63–76. [13] G. Sposito, The Surface Chemistry of Soils, Oxford University Press, New York, NY, 1984.
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329 [14] I.S. Shaban, F. Maca`sek, Influence of humic acids on sorption of cesium and strontium on montmorillonite, J. Radioanal. Nucl. Chem. 229 (1998) 73–78. [15] J.A. Hur, M. Schlautmana, Effects of pH and phosphate on the adsorptive fractionation of purified Aldrich humic acid on kaolinite and hematite, J. Colloid Interface Sci. 277 (2004) 264–270. [16] R. Zahn, C. von Schroetter, K. Wuthrich, Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding, FEBS Lett. 417 (1997) 400–404. [17] S. Lavie, G. Stotzky, Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of Histoplasma capsulatum, Appl. Environ. Microbiol. 51 (1986) 65–73. [18] G. Stotzky, H. Babich, Survival of, and genetic transfer by, genetically engineered bacteria in natural environments, Adv. Appl. Microbiol. 31 (1986) 93–138. [19] A.D. Gossert, S. Bonjour, D.A. Lysek, F. Fiorito, K. Wuthrich, Prion protein NMR structures of elk and of mouse/elk hybrids, Proc. Natl. Acad. Sci. USA 102 (2005) 646–650. [20] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a lamarckian
[21] [22] [23]
[24]
[25]
[26]
329
genetic algorithm and empirical free energy function, J. Comp. Chem. 19 (1998) 1639–1662. J.K. Mitchell, Fundamentals of Soil Behaviour, John Wiley & Sons, 1993. A. Piccolo, The supramolecular structure of humic substances, Soil Sci. 166 (2001) 810–832. L. Calzolai, D.A. Lysek, P. Guntert, C. von Schroetter, R. Riek, R. Zahn, K. Wuthrich, NMR structures of three single-residue variants of the human prion protein, Proc. Natl. Acad. Sci. USA 97 (2000) 8340–8345. E.M. Norstrom, J.A. Mastrianni, The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc, J. Virol. 80 (2006) 8521–8529. E.D. Belay, R.A. Maddox, E.S. Williams, M.W. Miller, P. Gambetti, L.B. Schonberger, Chronic wasting disease and potential transmission to humans, Emerg. Infect. Dis. 10 (2004) 977–984. M. Purdey, Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs? Med. Hypotheses 63 (2004) 211–225.
Biochemical and Biophysical Research Communications 367 (2008) 323–329 www.elsevier.com/locate/ybbrc
Organic polyanions act as complexants of prion protein in soil Maurizio Polano a,b, Claudio Anselmi a,c,*, Liviana Leita d, Alessandro Negro e, Maria De Nobili b a SISSA, Via Beirut 2-4, I-34014 Trieste, Italy Department of Agriculture and Environmental Sciences, Universita` di Udine, Via delle Scienze 208, I-33100, Italy c CNR-INFM-DEMOCRITOS, Via Beirut 2-4, I-34014 Trieste, Italy C.R.A., Centro di Ricerca per lo Studio delle Relazioni Pianta-Suolo, Sede Distaccata di Gorizia, Via Trieste 23, I-34170 Gorizia, Italy e CRIBI Biotechnology Centre, Universita` di Padova, Via U. Bassi 58/b, I-35131 Padova, Italy b
d
Received 13 December 2007 Available online 2 January 2008
Abstract The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity. 2007 Elsevier Inc. All rights reserved. Keywords: Chronic wasting disease; Humic acid; Infectivity of soil; Pollution of soil; Clay; Docking
Infectious proteinaceous particles causing transmissible spongiform encephalopathies (TSEs) have recently become matter of considerable environmental pollution and concern. TSEs are a class of fatal neurodegenerative disorders that affect animals and human beings, which include Creutzfeldt-Jakob disease in human beings, bovine spongiform encephalopathy in cattle, scrapie in sheep, and chronic wasting disease (CWD) in cervids. The ethiologic agent of TSEs is the prion, a proteinaceous particle composed mainly of PrPSc, the b-sheet-rich conformer of the cellular form of the prion protein (PrPC), a normal constituent of
Abbreviations: PrP, prion protein; MoPrP, recombinant murine prion protein; TSE, transmissible spongiform encephalopathy; CWD, chronic wasting disease; HS, humic substance; HA, humic acid; Mnt, montmorillonite. * Corresponding author. Address: SISSA, Via Beirut 2-4, I-34014 Trieste, Italy. Fax: +39 040 3787528. E-mail address: [email protected] (C. Anselmi). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.143
mammalian cells. Accumulation of PrPSc in the central nervous system of animals and humans leads to the manifestation of the disease [1]. PrPC is composed of about 250 amino acids, mainly in a-helical conformation and upon conversion to PrPSc, it is sufficient for the spontaneous formation of prions in mammals without the intervention of any exogenous agent [1]. This has been recently demonstrated with the production in vitro of the synthetic prion [2]. To date the diffusion of CWD in the natural environment is a matter of growing alarm in the USA. CWD was identified in 1977 as a TSE or prion disease, which appears to be freely transmitted among cervids by direct or indirect horizontal contact with infected biological material [3,4]. It seems possible, however, that under natural conditions prions can contaminate the environment through the soil. The scrapie infectious agent indeed persists in soil for years retaining its capability to activate misfolding of the native protein and infectivity [5]. Soil has been shown to retain the pathogenic agent for years in a
324
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
form that is experimentally transmissible to laboratory animals [6,7] and different soils have been shown to strongly retain prions [8]. The extent to which the prion is transmitted from a contaminated environment is unknown. Several articles have started to address the problem of prions in the soil [5,9– 11], but the present approaches show several drawbacks and technical difficulties, as soil is a complex, multi-component system of interacting materials, both mineral and organic. Most studies have so far been concerned with the adsorption capacity of clay minerals [9–11]. The protein appears to bind to the mineral surface with the permanent negative charges via electrostatic interactions and to undergo a pH independent conformational change in the adsorbed state, which leads to an increased b-sheet structure. No quantitative data on the adsorption capacity of the clay or on the effect of different cations on adsorption isotherms is available to date. In contrast, the contribution of soil organic components in adsorption has been comparatively neglected, as they are only a minor fraction on a weight basis. However, among organic molecules, humic substances (HSs) are natural polyanions that result among the most reactive compounds in the soil and possess the largest specific surface area [12]. Notably, the majority of mineral surfaces in soils and sediments are normally coated with organic matter [13] and the positively charged mineral surfaces are changed into negatively charged or their overall negative charge is enhanced [14]. Adsorption of HSs can modify the physicochemical properties of the underlying mineral surface, thus rendering hydrophilic mineral surfaces more hydrophobic and more capable of adsorbing organic contaminants [15]. In this work, we have studied the effect of HSs and ions on the adsorption of recombinant PrP onto different clay minerals. In fact, it is commonly admitted that recombinant PrP could serve as a model for the normal cellular prion protein PrPC [2]. Both experiments and theoretical modeling indicate that HSs participate in the adsorption of the PrP in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks with accessible polar residues. We show that HSs enhance the complexant capacity of soil, even if it is not clear if this raises or lower the prion infectivity. In contrast, our results suggest that studies that only focus on clays limit our knowledge about TSEs in the environment, because they completely neglect one of the most reactive components of soil and are therefore not representative of the actual situation.
Materials and methods Cloning of MoPrP. Full-length recombinant murine prion protein from 23 to 231 (MoPrP) was prepared in Escherichia coli according to a previously described strategy [16]. Concentration of MoPrP stock solutions was determined by UV absorbance spectroscopy, using a Varian (Carry 50) spectrophotometer and 1-cm path length quartz cuvettes. An extinction coefficient for MoPrP
of 2.7 mg 1 cm2 at 280 nm was calculated with the ProtParam tool on expasy (http://www.expasy.org). Preparation of clay suspensions. Clay minerals (Wyoming Na-montmorillonite (Na-Mnt) (SWy-1), Arizona Ca-montmorillonite (Ca-Mnt) (SAz-1) and kaolinite with a cation exchange capacity of 120, 76.4, and 9 c+ mol/kg, respectively) were suspended at a rate of 0.05 (Na-Mnt), 0.25 (Ca-Mnt) or 0.4 mg/mL (kaolinite) in either mQ water or PBS at different ionic strengths (1· and 0.1·). For kaolinite suspension, particles greater than 2 mm were allowed to settle out by gravity and discarded. Preparation of clay–HA complexes. 0.0122 g of HA (International Humic Substances Society (IHSS) peat reference IR103H-2) and 1 g kaolinite were suspended in 10 mL of solutions 1 mM NaOH and 2.5 mM CaCl2, respectively, and then mixed for about 2 h at room temperature. Suspension was then centrifuged at 4000 rpm for 5 min and dried at 40 C for 20 h. The dry material was ground to a fine powder and then the suspension 0.1405 mg/mL was prepared with mQ reagent grade water. Adsorption isotherms. Varying volumes of concentrated MoPrP stock solutions (about 35–37 lg/mL) were spiked into Eppendorf centrifuge tubes containing a fixed volume (0.2–0.5 mL) of mineral suspensions and a complementary volume of either mQ water or PBS to give a constant final volume of 2 mL at fixed solution chemistry conditions. Adsorption isotherms were measured in triplicate batch experiments. After 10 min shaking, the MoPrP/clay suspension was centrifuged for 95 min at 14,000g on an Eppendorf 5402 centrifuge. The amount of adsorbed protein was calculated by difference from the equilibrium concentration in solution (depletion method) [17]. Difference spectra were record using a Cary 50 spectrophotometer. Amounts adsorbed vs equilibrium concentrations were fitted to both the linearized form of the Freundlich equation and the Hanes–Woolf linearized form of the Langmuir equation [18]. Molecular docking. Taking into account the average molecular weight, charge density and the average number of functional groups of the standard HA adopted in the experiments, we designed a 3-D model molecule by randomly connecting the more common HA organic molecular fragments (Supplementary material). The automated docking procedure vs PrPC (PDB entry 1XYX) [19] was performed using the program AutoDock 3.0.5 [20]. One thousand different complexes were analyzed.
Results Equilibrium adsorption of MoPrP on clays Kaolinite and montmorillonite (Mnt) were chosen as model clays. Kaolinite is one of the most abundant minerals in soils and sediments. Mnt forms by crystallization from solutions high in soluble silica and magnesium and has a 2:1 layer structure in which Al3+ is the normal ion in the central octahedral sheet. However about one-eight of the octahedra contain Mg2+ and the surplus negative charge is neutralized by exchangeable cations adsorbed to the surface. Cohesion between sheets is not very strong and depends on the amount of water present. When wet conditions occur, water causes the clay to swell dramatically and internal negatively charged surfaces become accessible [21]. Fig. 1A shows the adsorption isotherms of MoPrP on different clay minerals. Different affinities for MoPrP are: Na-Mnt > CaMnt > kaolinite > quartz sand. The dependence of the binding capacity of the clay minerals can be explained on the basis of the different mineralogy of the clays, in particular it indicates the importance of both particle size and charge. Whereas kaolinite is made of relatively large non-
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
325
Fig. 1. (A) Adsorption isotherm of clays at pH 7.0 in water. X/M is the adsorbed amount of prion and Ce is the prion concentration in solution at equilibrium. (B) Adsorption isotherm of Na-Mnt in different buffer solutions. (C) Adsorption isotherm of MoPrP on pure kaolinite and on HA–kaolinite complex. (D) Adsorption isotherm of HA at pH 6.6 at different ionic strengths.
expanding crystals and therefore possesses a low number of charged absorption sites (as confirmed by Fig. 1A), Mnt consists of much smaller crystals, characterized by huge specific surface area (800 m3/g) and much higher negative charge density. The separation between individual platelets varies depending mainly on the type of interlayer cations present and the ionic strength of the surrounding solution. Na-Mnt and Ca-Mnt are expected to behave differently as saturation with a divalent cation does not allow the complete swelling of the clay. Fig. 1A shows how the adsorption capacity of Na-Mnt is higher than Ca-Mnt, according to the higher interlayer distance of the first with respect to the second. This enlightens the capacity of Na-Mnt of adsorbing the recombinant prion protein not only at the surface, but also inside the interlamellar space of the clay structure. In contrast, the adsorption on Ca-Mnt is likely to occur only on the external surface of the clay. Adsorbed MoPrP could not be removed from the clay, even by strongly denaturing solutions such as 1% SDS. Solution ionic strength can have an effect on the adsorption process because the interlayer is hydrated and cations within the interlayer may exchange with those in the external solution. In order to verify the dependence of the clay adsorption on the salt, we performed experiments using
either PBS buffer or a solution of NaCl with the same ionic strength. Fig. 1B shows that adsorption affinity of Na-Mnt vs MoPrP is lower when the clay is suspended in PBS with respect to water and to NaCl. Maximum adsorption capacity of the clay was observed in the NaCl solution (Fig. 1B). As PBS buffer contains NaCl, KCl, and Na2HPO4, it seems that both the affinity and the maximum amount of adsorption depend on the type of ions more than the actual ionic strength. In this case, it is likely that Na+/K+ exchange lowers clay expandability and makes internal surfaces less accessible. Effect of HSs Unfortunately, the exact composition of HSs is unknown, as they are complex mixtures of polyphenol–polycarboxilic acids with colloidal and supramolecular characteristics, which have never been separated into pure components [22]. Nevertheless, standard humic acids (HAs) can now be used to avoid the uncertainties due to their polydisperse nature and different extraction procedures. Fig. 1C shows that the kaolinite–HA complex greatly increases both the adsorption affinity and the maximum amount of absorbed protein with respect to pure kaolinite,
326
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
which weakly adsorbs prions (Fig. 1A). Notably, values for kaolinite–HA complex are in the same order of those of NaMnt, which is among the clays with the highest adsorption capacity (Fig. 1A and B). Also solid HA alone shows an enormous affinity for the prion protein at different ionic strengths (Fig. 1D), approximately twenty times that of Na-Mnt. Molecular interactions with HA in solution HSs cannot only be present in soil and sediments as solids, but are also dissolved in the soil solution or in surface waters. We examined the possibility of interactions of MoPrP with dissolved HA. The intense absorption band at 193 nm in the UV spectrum of MoPrP decreases linearly upon addition of HA (Fig. 2A and B) suggesting the occurrence of an interaction with the backbone of exposed amino acids. Complete titration is achieved at a HA/MoPrP ratio of about 4.8:1 (W/W) (Fig. 2A). It is also possible
that at larger HA/MoPrP ratios, humic molecules completely shield the protein preventing adsorption by chromophores. The absorbance at 228 nm increases upon addition of HA, reaching a maximum at an apparent molar ratio of about 1 (W/W) and declining thereafter for larger HA/ MoPrP ratios (Fig. 2B). The shoulder at 228 nm is generally attributed to the absorbance of Asp, Glu, Asn, Gln, Arg, and His side chains and to a weak transition of electrons in peptide bonds (210–230 nm). In the truncated form of MoPrP, exposed side chains of this types (>40% exposure) belong to the a1 helix (C class amino acids) and to the poorly structured region near the C-terminus (A class amino acids) [23]. Fig. 2B would be coherent with an increased solvent exposure of this type of side chains and in their subsequent shielding by humic molecules. The fact that the complete annealing of the adsorption at both 190 and 228 nm is reached for relatively close values of HA/ MoPrP supports a general shielding mechanism more than
Fig. 2. (A) Variation of the absorbance of MoPrP vs the concentration of HA. (B) Variation of the absorption at 228 nm of MoPrP at different concentration ratio with HA (W/W).
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
327
Fig. 3. Occurrence of PrPC amino acids at the interface with HA. Most relevant amino acids are labelled.
the binding to specific sites, whereas site specific interaction can occur at the beginning of the titration. Molecular docking In silico docking experiments were performed to characterize the nature of the interactions between HAs and
PrPC. HA can assume many different conformations and seem to bind PrPC without any apparent specificity. We therefore analyzed the docked complexes in order to find the amino acids that are more frequently at the interface with HA (Fig. 3), as it is supposed they could be more important for association. Due to the lack of binding specificity, practically all amino acids are interacting with HA
Fig. 4. (A) PrPC amino acids involved in the binding of HA. (B) Examples of PrPC–HA complexes; different HA conformations are colored in blue, red, yellow, orange, and green. (C) Representation of the electrostatic potential at the PrPC surface. Blue and red represents positive and negative values, respectively.
328
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329
in some complexes. In order to discriminate among true positives and the background noise, we evaluated frequency statistical dispersion and considered only those amino acids of which the number of counts differs from the mean value more than one standard deviation. They mostly correspond to two regions of PrPC, situated within helices a1 and a2, respectively (Figs. 3 and 4A and B), which correspond to zones that have been suggested to be sensible in the process of conversion of PrPC toward the pathogenic form [24]. Notably, also UV absorption spectra suggest the occurrence of conformational changes of residues within helix a1 upon interactions with HA. Analysis of PrPC surface shows that electrostatics plays a major role in stabilizing PrPC–HA complexes, as interface regions corresponds to two maxima of the electrostatic potential (Fig. 4C). However, also polar residues play a major role in binding. In particular, Asn side chains are both donors and acceptors of hydrogen bonds and N171, N173, N174, and N181 at helix a2 are among the amino acids that more frequently bind HA (Fig. 3). Discussion Several studies have demonstrated prions can be retained in soil in a form that is able to cause the insurgence of TSEs even several years after the soil contamination. However, within the large spread of potentially infected lands, prion diseases have become endemic only in geographically limited regions [25]. Reasons for this remain unknown, but it suggests a role of the different kinds of soil in either enhancing or containing prion infectivity. In fact, as the latter depends on a conformational change of the prion itself, interactions with substances able to enhance the change could favor infectivity. In this context, a good knowledge of the mechanisms of retention of prion in soils is of great importance for controlling the possibilities of dissemination of TSEs. Several aspects play a role in this process, such as the efficiency of sorption and desorption of the prion in soil and its capacity to undergo conformational changes and/or degradation. Our results suggest that MoPrP adsorption in soil is driven both by HAs and clays. The nature of the clay seems to be important as well as that of the ions in solution, as it is likely they influence clay expandability and internal surface accessibility to the protein. This suggests that different types of soil with different clays and ions could involve different fates for the prion. However, HAs not only show the strongest affinity for MoPrP, but they also considerably enhance the adsorption capacity of clays. Our results provide evidence of direct interactions between HAs and prions and suggest HAs promote conformational changes in some prion residues, which are located in regions of relevance for the conversion to the pathogenic form. There is further evidence in the literature that polyanionic molecules induce an efficient conversion of PrPC in vitro, eventually enhancing fibril growth. In contrast, recent studies
seem to exclude the possibility of a a–b conversion from a non-pathogenic form of prion to a pathogenic form induced by the interaction with soil clays [9]. On the other hand, low contents of soil organic matter is often associated to a number of CWD cases among free-ranging cervids in USA [25], whereas other studies suggest that factors, as metal ions, which are known to sequestrate HSs in soils, promote the insurgence of CWD [26]. Of course, biological tests on animals are needed to elucidate whether interaction with HSs can lead to either protection or deactivation of prion infectivity in soil. Acknowledgments Authors thank Prof. Giuseppe Legname, Prof. Paolo Carloni (SISSA), and Prof. Catia Sorgato (University of Padua) for valuable advices. This work was carried out with a grant of the Ministry of Agricultural Policies, finalized project RIFAFERT. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 12.143. References [1] S.B. Prusiner, Early evidence that a protease-resistant protein is an active component of the infectious prion, Cell 116 (2004) S109–S111. [2] G. Legname, I.V. Baskakov, H.O. Nguyen, D. Riesner, F.E. Cohen, S.J. DeArmond, S.B. Prusiner, Synthetic mammalian prions, Science 305 (2004) 673–676. [3] E.S. Williams, S. Young, Spongiform encephalopathies in Cervidae, Rev. Sci. Tech. 11 (1992) 551–567. [4] E.S. Williams, M.W. Miller, Chronic wasting disease in deer and elk in North America, Rev. Sci. Tech. 21 (2002) 305–316. [5] B. Seidel, A. Thomzig, A. Buschmann, M.H. Groschup, R. Peters, M. Beekes, K. Terytze, Scrapie agent (strain 263K) can transmit disease via the oral route after persistence in soil over years, PLoS ONE 2 (2007) e435. [6] P. Brown, D.C. Gajdusek, Survival of scrapie virus after 3 years’ interment, Lancet 337 (1991) 269–270. [7] D.M. Taylor, Resistance of transmissible spongiform encephalopathy agents to decontamination, Contrib. Microbiol. 11 (2004) 136–145. [8] R.C. Angers, S.R. Browning, T.S. Seward, C.J. Sigurdson, M.W. Miller, E.A. Hoover, G.C. Telling, Prions in skeletal muscles of deer with chronic wasting disease, Science 311 (2006) 1117. [9] M. Revault, H. Quiquampoix, M.H. Baron, S. Noinville, Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays, Biochim. Biophys. Acta 1724 (2005) 367–374. [10] C.J. Johnson, K.E. Phillips, P.T. Schramm, D. McKenzie, J.M. Aiken, J.A. Pedersen, Prions adhere to soil minerals and remain infectious, PLoS Pathog. 2 (2006) e32. [11] L. Leita, F. Fornasier, M. De Nobili, A. Bertoli, S. Genovesi, P. Sequi, Interactions of prion proteins with soil, Soil Biol. Biochem. 8 (2006) 1638–1644. [12] N. Senesi, Binding mechanisms of pesticides to soil humic substances, Sci. Total Environ. 123–124 (1992) 63–76. [13] G. Sposito, The Surface Chemistry of Soils, Oxford University Press, New York, NY, 1984.
M. Polano et al. / Biochemical and Biophysical Research Communications 367 (2008) 323–329 [14] I.S. Shaban, F. Maca`sek, Influence of humic acids on sorption of cesium and strontium on montmorillonite, J. Radioanal. Nucl. Chem. 229 (1998) 73–78. [15] J.A. Hur, M. Schlautmana, Effects of pH and phosphate on the adsorptive fractionation of purified Aldrich humic acid on kaolinite and hematite, J. Colloid Interface Sci. 277 (2004) 264–270. [16] R. Zahn, C. von Schroetter, K. Wuthrich, Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding, FEBS Lett. 417 (1997) 400–404. [17] S. Lavie, G. Stotzky, Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of Histoplasma capsulatum, Appl. Environ. Microbiol. 51 (1986) 65–73. [18] G. Stotzky, H. Babich, Survival of, and genetic transfer by, genetically engineered bacteria in natural environments, Adv. Appl. Microbiol. 31 (1986) 93–138. [19] A.D. Gossert, S. Bonjour, D.A. Lysek, F. Fiorito, K. Wuthrich, Prion protein NMR structures of elk and of mouse/elk hybrids, Proc. Natl. Acad. Sci. USA 102 (2005) 646–650. [20] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a lamarckian
[21] [22] [23]
[24]
[25]
[26]
329
genetic algorithm and empirical free energy function, J. Comp. Chem. 19 (1998) 1639–1662. J.K. Mitchell, Fundamentals of Soil Behaviour, John Wiley & Sons, 1993. A. Piccolo, The supramolecular structure of humic substances, Soil Sci. 166 (2001) 810–832. L. Calzolai, D.A. Lysek, P. Guntert, C. von Schroetter, R. Riek, R. Zahn, K. Wuthrich, NMR structures of three single-residue variants of the human prion protein, Proc. Natl. Acad. Sci. USA 97 (2000) 8340–8345. E.M. Norstrom, J.A. Mastrianni, The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc, J. Virol. 80 (2006) 8521–8529. E.D. Belay, R.A. Maddox, E.S. Williams, M.W. Miller, P. Gambetti, L.B. Schonberger, Chronic wasting disease and potential transmission to humans, Emerg. Infect. Dis. 10 (2004) 977–984. M. Purdey, Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs? Med. Hypotheses 63 (2004) 211–225.