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Lipopolysaccharide induced conversion of recombinant prion protein a
b
bc
b
Fozia Saleem , Trent C Bjorndahl , Carol L Ladner , Rolando Perez-Pineiro , Burim N d
Ametaj & David S Wishart
abc
a
Department of Biological Sciences; University of Alberta; Edmonton, AB Canada
b
Department of Computing Science; University of Alberta; Edmonton, AB Canada
c
National Institute for Nanotechnology; Edmonton, AB Canada
d
Department of Agricultural, Food and Nutritional Science; University of Alberta; Edmonton, AB Canada Published online: 12 May 2014.
Click for updates To cite this article: Fozia Saleem, Trent C Bjorndahl, Carol L Ladner, Rolando Perez-Pineiro, Burim N Ametaj & David S Wishart (2014) Lipopolysaccharide induced conversion of recombinant prion protein, Prion, 8:2, 221-232, DOI: 10.4161/ pri.28939 To link to this article: http://dx.doi.org/10.4161/pri.28939
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Research Paper Prion 8:2, 221–232; March/April 2014; © 2014 Landes Bioscience
Lipopolysaccharide induced conversion of recombinant prion protein Fozia Saleem1, Trent C Bjorndahl2, Carol L Ladner2,3, Rolando Perez-Pineiro2, Burim N Ametaj4, and David S Wishart1,2,3,* 3
1 Department of Biological Sciences; University of Alberta; Edmonton, AB Canada; 2Department of Computing Science; University of Alberta; Edmonton, AB Canada; National Institute for Nanotechnology; Edmonton, AB Canada; 4Department of Agricultural, Food and Nutritional Science; University of Alberta; Edmonton, AB Canada
Abbreviations: TSE, transmissible spongiform encephalopathy; CJD, Creutzfeldt Jakob disease; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; PMCA, Protein Misfolding Cyclic Amplification; EM, electron microscopy; TEM, transmission electron microscopy; PK, proteinase K; PrP, prion protein; ShPrP, the core alpha helical Syrian hamster prion protein residues 90-232; ShPrPβ, beta-sheet converted Syrian hamster prion protein (90-232); PrPC, cellular prion protein; LPS, lipopolysaccharide; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol); SDS, sodium dodecyl sulfate; PE, phosphatidylethanolamine; DOPE, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine; PMSF, phenylmethanesulfonyl fluoride; CMC, critical micelle concentration; CD, circular dichroism; NMR, nuclear magnetic resonance
The conformational conversion of the cellular prion protein (PrPC) to the β-rich infectious isoform PrPSc is considered a critical and central feature in prion pathology. Although PrPSc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrPC to proteinase K resistant PrPSc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrPC conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrPC to PrPSc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).
Introduction Prion diseases or transmissible spongiform encephalopathies (TSEs), including Kuru, variant Creutzfeldt Jakob disease (vCJD), bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), and scrapie are all examples of incurable and uniformly fatal neuro-degenerative diseases that arise from the self-propagation of misfolded prion proteins. The current working hypothesis is that small numbers of misfolded, β-rich prions (called PrPSc) are able to catalytically convert native, monomeric, helix-rich cellular prion protein (PrPC ) to large numbers of misfolded prions,1 which aggregate into larger, uniformly organized, oligomeric substructures that can further assemble into fibrillar, macrostructures. In this way, the misfolded prions are able to spread exponentially leading to loss of neuronal function wherever PrPC is highly expressed. PrPC is found in all mammalian cells; but the highest level of expression is in neurological tissues,2,3 which largely explain the neurodegenerative nature of prion diseases.
The native prion protein, PrPC, is a lipid-bound, monomeric, ~24 kDa protein with an unstructured N-terminal region containing roughly 70 residues and a globular core consisting primarily of α-helical secondary structure4 (42% α-helix and about 5% β-sheet). This unstructured N-terminus consists of 5 octa-repeat segments that have been shown to bind divalent metal ions (Cu2+, Fe2+, Zn2+).5,6 The core contains 2 N-linked glycosylation sites at Asn181 and Asn1977,8 while the C-terminus is linked to a lipid glycosylphosphatidylinositol (GPI) anchor that secures the protein to the cellular membrane. PrPSc is rich in β-sheet structure that contains 10% α-helix and 30% β-sheet.1,9,10 However, a recent study showed that PrPSc consists largely of β-strands with no α-helices.11 These β-sheets tends to form large oligomers, insoluble aggregates, and fibril like structures. As a result, PrPSc is protease-resistant, oligomeric, and substantially less soluble than PrPC. These divergent features allow the 2 isoforms to be easily differentiated biochemically. After many decades of investigation, much is known of the prion protein’s (PrPC) native structure; however, less is known about the
*Correspondence to: David S Wishart; Email: [email protected] Submitted: 01/01/2014; Revised: 04/10/2014; Accepted: 04/16/2014; Published Online: 05/12/2014 http://dx.doi.org/10.4161/pri.28939 www.landesbioscience.com Prion 221
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Keywords: prion protein, protein misfolding, lipopolysaccharide, beta oligomer, fibril
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continued exposure to detergents. Despite the widespread belief in the “protein-only” hypothesis concerning prion protein conversion and infectivity, controversies over whether other molecules beside PrPSc are also required for conversion and replication in vivo, still remain. In particular, the role of a number of naturally occurring, non-proteinaceous molecules such as nucleic acids,19 polyanions,15,20 lipids,21-23 sulfated glycans,15 and metals24 are actively being investigated in the pathogenesis of prion diseases. Recently, it was shown that recombinant prion proteins could be rendered seed-competent (and infectious) with the addition of total liver RNA and synthetic 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) lipid molecules.20 Even more recently it was reported that phosphatidylethanolamine (PE) is an essential cofactor for generating infectivity from converted recombinant PrP.25 These findings strongly suggest that a non-protein molecular component may be responsible for facilitating or augmenting the prion protein conversion process.19,25 Given these emerging results, we began screening for a “prion protein converting molecule” among naturally occurring, exogenous molecules. The recent discovery that the N-terminal residues of the human prion protein (PrPC ) has broad spectrum antibacterial properties26 mediated by membrane-prion protein interactions directed our screen toward analyzing bacterial components and specifically looking at lipopolysaccharide (LPS), an outer membrane component of gram-negative bacteria. In particular, LPS is a temperature resistant, amphipathic molecule that consists of both lipid (lipid A) and carbohydrate (core and O-antigen) components. LPS acts as an immunogen and a strong pyrogen in healthy mammals. It binds the CD14/ Figure 1. Region of the 15N-HSQC NMR spectra for the ShPrP90–232 TLR4/MD2 receptor complex which resides in lipid rafts before (A) and after (B) the addition of an equimolar amount of LPS. where the prion protein co-localizes.27 LPS promotes the secretion of pro-inflammatory cytokines in many cell β rich isoform. Likewise, very little is known about the α-helix to types, but especially in macrophages. Given that both β-sheet conversion process, the precise mechanisms behind self- PrPC and the LPS/CD14/TLR4 receptor complex are localized seeded propagation or the potential causative agents that might to the same membrane domains via their GPI anchors and given induce sporadic prion disease. As prion protein misfolding and the structural similarity of LPS to polyanionic molecules like aggregation is believed to be a major contributor to prion disease POPG that have been associated with prion protein conversion, etiology, a more detailed molecular understanding of the ordered we decided to investigate LPS’s potential effects on ShPrP (90– aggregation and amyloid fibril formation process is critical for 232) in vitro. developing strategies to prevent or treat these conditions. Interestingly, we found that native, recombinant prion To help better understand biochemical aspects of PrP protein incubated with modest concentrations of LPS (15 μg/ conversion, several groups have used in vitro or test-tube systems mL) at physiological normal pH and temperature led to the in which recombinant PrPC molecules are converted into to β-rich rapid conversion to oligomers that were PK resistant and rich in PrPSc-like molecules (PrPβ) using a number of chemical and/or β-sheet structure (PrPβ) compared with the native ShPrP (90– physical agents such as guanidine hydrochloride,12 urea,13 SDS,14 232). Furthermore this conformational conversion to a β-sheet high temperature,15 low pH16 that cause complete or partial rich structure occurs at ratios of PrP to LPS below 1:1 (w/w), as protein denaturation. Most of these protocols not only cause long as the LPS concentration in the conversion solution remains the conformational change of PrPC into PrPβ but also generate above the critical micelle concentration (CMC). Even more “synthetic prions” that exhibit many of the physiochemical interestingly, this conversion phenomenon, whereby conversion to properties seen in PrPSc molecules isolated from diseased tissues.17,18 a β-sheet rich structure occurred down to a weight ratio of ShPrP However, unlike brain-derived prions, the conversion and to LPS of 1:0.09 (w/w), was not seen with other known prion apparent propagation of these synthetic prions is dependent on conversion reagents (including SDS, urea, POPG, or PE). Given
the ubiquity of LPS in both recombinant prion preparations and the abundance of LPS in many animal pens, cages, and feed, these results may have some interesting implications regarding prion conversion – both in vitro and in vivo.
Results LPS mediated conversion The strong interaction between the prion protein and bacterial LPS was evident from our initial binding studies performed by NMR (Fig. 1). Apart from a few N-terminal and 6x-His affinity tag residues, immediate and complete attenuation of ShPrP (90–232) 15N-HSQC amide signals resulted upon addition of a 1:1 ratio (w/w) of LPS to the ShPrP (90–232) sample. This interaction was also visualized by transmission electron microscopy (Fig. 2). As seen in Figure 2, the typical cylindrical bicellular structure adopted by LPS in water was disrupted leading to a spherical micelle formation (Fig. 2B) with micelle diameters ranging between 50 and 150 nanometers. The negative staining achieved with uranyl acetate suggests that protein is adhering to the micelle surface. More interestingly, conversion to a fibril state was observed upon incubation of this complex at 37 °C (Fig. 2C and D). These converted prion protein fibrils ranged in length from 100 nm to 1 μm and were associated in pairs and clustered with the LPS micelles. To further characterize this state, a circular dichroism
(CD) spectrum was collected (Fig. 3B) and a proteinase K (PK) digestion assay was performed (Fig. 3A). The CD spectrum revealed a high level of β-sheet structure and the PK digestion revealed a 12 kDa proteolytic resistant core, consistent with both β-oligomeric28 and spontaneous PMCA-derived isoforms.29 To test if this phenomenon was restricted to Syrian hamster prion protein we also found that mixing pure LPS in the presence of NaCl (150 mM) with recombinant mouse prion protein at mg ratios (w/w) of 1:1 (LPS:ShPrP (90–232) also resulted in instant conversion to PrPβ (result not shown). These preliminary findings warranted a more in-depth characterization of the LPS mediated conversion process. In particular we decided to investigate the effect of differing LPS concentrations on the prion conversion process. Figure 4 shows the results of the LPS-mediated conversion in which solutions with progressively lower ShPrP (90–232) to LPS ratios were prepared and monitored by CD. Weight (w/w) ratios above 1:0.5 (ShPrP (90–232):LPS) resulted in the complete conformational change of PrPC into β-sheet oligomers. Meanwhile, solutions in which the LPS was below the critical micelle concentration (CMC) of 14 μg/mL did not show any conversion at all, even after 4 wk of incubation. Thus we conclude that a micellar form of LPS (as might be found in a live or lysed bacterial cell) is required for initiating conversion. The secondary structure content (% α-helix and β-sheet) of the prion proteins were calculated from the CD data presented in Figure 4 for each dilution and are shown in Table 1.
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Figure 2. Electron microscope images of the LPS mediated conversion of the ShPrP (90–232) protein. (A) LPS micelles at 0.5 mg/mL in water, pH 7.0 (magnification 71 000×, scale bar 50 nm). (B) The same LPS “micelles” shown in (A) after addition of 0.5mg/mL ShPrP (magnification 56 000×, scale bar 200 nm). (C and D) The sample after the addition of 150 mM NaCl, 0.1% NaN3 and incubated at 37 °C for 48 h magnification 110 000× and 14 000×, scale bars: 50 nm and 0.5 um respectively). Inset in Figure 2D was collected at 110 000× (scale bar 200 nm). Small fibrils can be seen in C. Samples were stained with lead acetate (A) or uranyl acetate (B–D).
(with PrPβ) was serially diluted by half, by adding equal volumes of fresh recombinant ShPrP (90–232) of the same concentration to the LPS converted PrPβ. This serial dilution process generated subsequent solutions with ShPrP (90–232) to LPS ratios (w/w) of 1:6, 1:3, 1:1.5, 1:0.75, 1:0.38, 1:0.17, 1:0.09, and 1:0.01 (Fig. 4; Table 1). Interestingly conversion of ShPrP (90–232) to a β-sheet rich structure (above 20% β-sheet) was maintained even when the molar ratio (mol/mol) of LPS to ShPrP (90–232) fell below 1:1 (equivalent to 1:0.38 w/w ratio of ShPrP (90–232):LPS). This is unlike other conversion methods that only showed a dilution effect upon addition of fresh ShPrP (90–232). This confirmed that a direct interaction and conversion was being observed between LPS and the protein. It also confirmed that the measured conformational change was not simply due to a denaturation effect. This conversion was observable at all LPS concentrations, until the LPS concentration dropped below its CMC (ShPrP (90– 232):LPS ratio (w/w) of 1:0.01). This effect is likely due Figure 4. CD spectra for 25 μM of ShPrP (90–232) with various concentration (w/w to the sensitivity of the experimental method used to ratios) of LPS (1:6 300 μM LPS, 1: 3–150 μM LPS, 1:1.5–75 μM LPS, 1:0.75–37.5 μM detect the change. More importantly, this serial dilution LPS, 1:0.38–18.5 μM LPS, 1:0.17–9.5 μM LPS, 1: 0.09–5 μM LPS, and 1:0.01–1 μM effect was used with RENAGE gel analysis (discussed LPS). Spectra were collected immediately after adding LPS, except for the 1:0.01 below) to ascertain the molecular interaction ratio. w/w ratio of LPS with shPrP(90–232), which was incubated an additional 4 wk to ascertain any changes in secondary structure. LPS-mediated PrPβ conversion and its subsequent serial dilution was compared with the conversion and Serial dilution serial dilution mediated by other chemical denaturants: Solutions containing LPS, unconverted recombinant ShPrP namely SDS, POPG, DOPE, and urea (Fig. 5; Table 2). While (90–232), and LPS-converted PrPβ (in various concentrations) LPS, SDS, POPG, and PE all share lipid moieties, only PE has a was also used to assess the unique concentration dependence positive charge or a cationic group provided by a primary amine. property of LPS in PrP conversion. Mixing pure LPS in the Furthermore, while SDS and POPG both contain negatively presence of NaCl (150 mM) with recombinant ShPrP (90– charged polar heads only LPS contains a phosphate group along 232) at a ratio (w/w) of 1:6 (ShPrP (90–232):LPS) resulted in with a complex saccharide. LPS-generated PrPβ demonstrates a instantaneous conversion to a PrPβ isoform (measured by the capacity to convert prion protein even at sub molar ratios as the percentage of helical and β-sheet structure determined by CD). concentration of LPS is reduced (down to its CMC) in contrast After confirming the conversion, the LPS-containing solution to all other reagents which show a progressive dilution effect. In
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Figure 3. (A) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrPβ . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the right). Lane 5: PK:ShPrPβ 1:1000. Lane 6: PK:ShPrPβ 1:750. Lane 7: PK:ShPrPβ 1:500. Lane 8: PK:ShPrPβ 1:250. (B) CD spectra for the ShPrP (90–232) with 1:1 w/w ratio of LPS at 0 incubation time.
Table 1. Secondary structure content (measured by CD) of LPS-converted prions α-Helix (%)
β-Sheet (%)
42
9
9
32
ShPrP (90–232): LPS (1:3)
10
31
ShPrP (90–232): LPS (1:1.5)
8
33
ShPrP (90–232): LPS (1:0.75)
12
31
ShPrP (90–232): LPS (1:0.38)
17
29
ShPrP (90–232): LPS (1:0.17)
20
24
ShPrP (90–232): LPS (1:0.09)
26
20
ShPrP (90–232): LPS (1:0.01)
41
10
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ShPrP (90–232) control ShPrP (90–232): LPS (1:6)
Figure 5. CD spectra for the ShPrP (90–232) with various molar ratios of DOPE, SDS, POPG, and urea at 0 incubation time. (A) CD spectra of the prion propagation studies using DOPE (diluted from 1:64 to 1:16 PrPC:DOPE (w/w)). (B) Urea induced oligomers and fibrils (for comparison of secondary structure content). (C) SDS (1:0.2 to 1:0.0125 PrPC:SDS). D. POPG (diluted from 1:0.5 to 1:0.065 of ShPrP [90–232]:POPG). Each diluted sample was incubated an additional 19 d to ascertain any changes in secondary structure.
other words, as the concentration or urea, POPG, and SDS is reduced, the extent of conversion is reduced. Interestingly SDS can only convert ShPrP (90–232) to PrPβ at concentrations
below its CMC of 8 mM (0.23% w/v). On the other hand conversion with urea and salt is time dependent with a faster conversion below the denaturation midpoint of 3.6 M.30 This
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Sample (condition)
Table 2. Secondary structure content of urea, DOPE, POPG, and SDS mediated conversion of prion protein DOPE induced conversion
*Urea induced conversion
Sample
α-Helix (%)
β-Sheet (%)
Sample
α-Helix (%)
β-Sheet (%)
ShPrP: DOPE (1:64)
41
10
ShPrP Native
43
11
ShPrP: DOPE (1:32)
40
12
Urea-induced ShPrP Oligomers
14
23
ShPrP: DOPE (1:16)
40
13
Urea-induced ShPrP Fibrils
10
27
POPG induced conversion
Sample
α-Helix (%)
β-Sheet (%)
Sample
α-Helix (%)
β-Sheet (%)
ShPrP: SDS (1:0.2)
13
31
ShPrP: POPG (1:0.5)
12
30
ShPrP: SDS (1:0.1)
27
18
ShPrP: POPG (1:0.25)
35
14
ShPrP: SDS (1:0.05)
38
13
ShPrP: POPG (1:0.125)
38
12
ShPrP: SDS (1:0.025)
40
10
ShPrP: POPG (1:0.0625)
42
8
ShPrP: SDS (1:0.0125)
42
8
Each diluted sample was incubated an additional 19 d to ascertain any changes in secondary structure. *Urea induced conversion: secondary structure values calculated for urea/guanidine converted β oligomers and fibrils used to compare with secondary structure values of controls (DOPE,SDS, and POPG) and LPS converted ShPrP.
suggests the importance of a partially unfolded intermediate in urea induced conversion. POPG has been shown to facilitate the conversion of recombinant PrP into an infectious strain capable of propagating in mice.20 POPG contains 2 alkyl carbon chains attached to a dihydroxylated phosphate group. Like LPS, it requires concentrations above its CMC (and therefore in lipid vesicles) to convert recombinant ShPrP (90–232) to PrPβ.31 However, unlike LPS, the extent of POPG conversion appears to be directly proportional to the concentration of POPG used (Fig. 5D), as the concentration of POPG is reduced, the extent of conversion is largely reduced. Quite recently it was also reported that phosphatidylethanolamine (PE or DOPE) can act as a solitary cofactor in the propagation of recombinant PrPSc using brain homogenate and standard PMCA conditions.25,32 To explore this further, we tested whether the same concentrations of DOPE described in the original publication25 could facilitate the conversion of ShPrP (90–232) to a β-sheet conformation without brain homogenate and sonication. Interestingly, our results showed that no conversion to PrPβ occurred, even after 7 d of incubation at 37 °C (Fig. 5A). These findings suggest that other factors, such as temperature or additional brain homogenate cofactors are responsible for initiating the conversion process mediated by PE. Given that the previous propagation studies of both DOPE and POPG were used in conjunction with other cellular components (cell extracts, RNA), it suggests that additional agents are required to mediate the conversion of PrPC to a propagating isoform. Our findings suggest that saccharide moieties (which are found only in LPS) may have the necessary requirements to facilitate conformational conversion. Size comparison of PrPβ formed by conversion and fibril propagation To compare the size of PrPβ aggregates formed by the various prion protein conversion techniques we used a native gel electrophoresis technique called RENAGE.33 With RENAGE it has been previously shown that the PrPβ oligomers formed via
urea/salt conversion consist of a distribution of heptamers to dodecamers.33 This size distribution of medium-sized oligomers is seen in Figure 6. In contrast, LPS-converted prion protein (PrPβ) are characterized by very large aggregates that migrate just a few millimeters into the stacking gel. The large size of these oligomers may be due to the presence of tightly bound LPS. Quantifying the protein bands for the LPS-converted prion proteins at 1:0.09 PrP:LPS shows that 19% of PrP is found in the large aggregate band, 15% is found in oligomers, 6% is in the dimer band and the rest is in the monomer band. Given that the molecular weight of monomeric LPS is roughly half that of the prion protein, the 1:0.09 weight (w/w) ratio equates to a 1:0.18 molar (mol/mol) ratio. This correlates strongly with the 19% protein band shown in RENAGE. An equimolar component of the protein is found in the oligomers. We propose that these oligomers are loosely associated with the tightly bound protein. More importantly, this loosely bound protein, while dissociable from the micellar bound component, still persists in a β-sheet rich, oligomeric state. Based on a two-state model, the native α-helical prion protein (ShPrP (90–232) has an α helix content of 42% and β-sheet content 9% while the LPSconverted PrPβ has only 10% α helix and 32% β-sheet structure (Table 1). If only the oligomeric, LPS-bound 18% of protein was in fact converted, the resulting CD spectra of the 2 states should present CD spectra showing 36% helix and 14% β sheet. Instead, a calculated CD spectra for 40% converted PrPβ protein and 60% unconverted (ShPrP [90–232]) monomer would result in a CD spectra with 29% α helix and 18% β sheet composition, which is close to the observed values (Table 1). Thus, twice the amount of β-sheet structure in LPS-converted PrPβ than predicted for a 1:0.18 ShPrP (90–232):LPS molar ratio, indicates a stoichiometric interaction ratio of 2:1 ShPrP (90–232):LPS. RENAGE was also used to compare oligomer sizes and structure induced by POPG and SDS. As seen in Figure 6, RENAGE of the POPG-converted prion protein (PrPβ ) shows only a small amount of aggregation. The POPG-converted oligomers appear
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SDS induced conversion
Figure 6. Resolution enhanced native acidic gel electrophoresis (RENAGE) shows different size and stability of oligomers formed from the different conversion methods. Conversion of recombinant ShPrP (90–232) is compared for urea/salt conversion (3M urea 20 mM sodium acetate pH 4 and 200 mM NaCl), POPG conversion (0.05:1 POPG to PrP [w/w], incubated at 37 °C for 4 d), SDS conversion (0.02% SDS), and LPS conversion (1:0.09 PrP to LPS, incubated at 37 °C for 2 d), 8 μg of each sample is loaded. The latter is from MoPrP (90–231) cross-linked nonspecifically using PICUP.
initiating the PrP conversion reactions and that nucleic acids do not mediate the conversion or influence the LPS-directed conversion.
Discussion In this study we have shown that LPS interacts with recombinant prion protein and that this interaction converts the α-helix-rich recombinant ShPrP (90–232) into a β-rich, PK-resistant form (PrPβ) under physiological conditions. LPS induced PrPβ conversion does not require any brainderived PrPβ seed, additional co-factors, or denaturants. This is in marked contrast to most other conversion and propagation protocols.14,16,20,29,38,39 Furthermore, many published conformational prion protein conversion strategies rely on a molar excess or destabilizing amounts of chemical denaturants (urea, guanidine hydrochloride), synthetic detergents (SDS), or extremes of pH or high (denaturing) temperatures.12-16 However, LPS-mediated prion protein conversion is distinct from these methods in that measurable conversion to a β-sheet rich form occurs at ShPrP (90–232) to LPS ratios down to 1:0.09 (w/w). Additionally, sizing of oligomers using RENAGE shows that the LPS converted PrPβ oligomers differ from those generated by POPG, SDS, and urea. Furthermore, RENAGE data showed that the LPS-generated PrPβ oligomers will dissociate to monomers. This differs from PrPβ oligomers formed by urea (Fig. 6) or acid,33 which are stable under electrophoresis conditions.
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to be unstable under native electrophoresis. Similarly, conversion of ShPrP (90–232) with 0.02% w/v SDS resulted in large aggregates that appear to dissociate as electrophoresis proceeds. While both the SDS and POPG conversion techniques produced β-rich oligomers with about 30% β sheet content (Fig. 6), only the POPG formed oligomers could dissociate into a monomeric state, while the SDS formed PrPβ remained in a loosely associated oligomeric state. Efforts to generate PrPβ oligomers or fibrils using DOPE were not successful and so they were not studied by RENAGE. In contrast, the LPS generated PrPβ is distributed into various states (Fig. 6). We also found that mixing LPS with recombinant cervid prion protein or recombinant mouse prion protein at a weight ratio of 1:1 LPS:ShPrP (90–232) (w/w) also resulted in conversion to large aggregates (result not shown). Nucleic acid control experiments Discrepancies exist in the literature regarding the necessary components required to induce conversion of the prion protein into a self-replicating form.15,20,23,34,35 While cofactors are not necessary for converting recPrPC to PrPSc in prion seeded PMCA,36 cellular components like RNA and lipids are required to generate de novo, self-propagating prions from recombinant prion protein sources.19,20 This issue needed to be addressed given that the commercial sources of LPS are contaminated with various amounts of nucleic acids (RNA, DNA, short oligos, and free NTPs). To rule out the potential effects that certain bacterial cellular components from E. coli might have on our observed reactions we sought to control for these known LPS contaminants. While RNA is reported to interact with N-terminal residues of PrP,35,37 DNA has been shown to interact with the C-terminal residues of the prion protein.34 Given that our recombinant prion protein construct is devoid of any reported RNA binding site and that our lab does not control for RNase activity, the influence that RNA might have on the observed conversion was of less concern than that of DNA. Two sources of LPS were obtained from Sigma. One preparation (L3012) is purified by gel filtration and is reported to have >10% nucleic acid contamination, while the other (L3024) is further purified by ion-exchange chromatography and contains
Prion Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kprn20
Lipopolysaccharide induced conversion of recombinant prion protein a
b
bc
b
Fozia Saleem , Trent C Bjorndahl , Carol L Ladner , Rolando Perez-Pineiro , Burim N d
Ametaj & David S Wishart
abc
a
Department of Biological Sciences; University of Alberta; Edmonton, AB Canada
b
Department of Computing Science; University of Alberta; Edmonton, AB Canada
c
National Institute for Nanotechnology; Edmonton, AB Canada
d
Department of Agricultural, Food and Nutritional Science; University of Alberta; Edmonton, AB Canada Published online: 12 May 2014.
Click for updates To cite this article: Fozia Saleem, Trent C Bjorndahl, Carol L Ladner, Rolando Perez-Pineiro, Burim N Ametaj & David S Wishart (2014) Lipopolysaccharide induced conversion of recombinant prion protein, Prion, 8:2, 221-232, DOI: 10.4161/ pri.28939 To link to this article: http://dx.doi.org/10.4161/pri.28939
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Research Paper Prion 8:2, 221–232; March/April 2014; © 2014 Landes Bioscience
Lipopolysaccharide induced conversion of recombinant prion protein Fozia Saleem1, Trent C Bjorndahl2, Carol L Ladner2,3, Rolando Perez-Pineiro2, Burim N Ametaj4, and David S Wishart1,2,3,* 3
1 Department of Biological Sciences; University of Alberta; Edmonton, AB Canada; 2Department of Computing Science; University of Alberta; Edmonton, AB Canada; National Institute for Nanotechnology; Edmonton, AB Canada; 4Department of Agricultural, Food and Nutritional Science; University of Alberta; Edmonton, AB Canada
Abbreviations: TSE, transmissible spongiform encephalopathy; CJD, Creutzfeldt Jakob disease; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; PMCA, Protein Misfolding Cyclic Amplification; EM, electron microscopy; TEM, transmission electron microscopy; PK, proteinase K; PrP, prion protein; ShPrP, the core alpha helical Syrian hamster prion protein residues 90-232; ShPrPβ, beta-sheet converted Syrian hamster prion protein (90-232); PrPC, cellular prion protein; LPS, lipopolysaccharide; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol); SDS, sodium dodecyl sulfate; PE, phosphatidylethanolamine; DOPE, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine; PMSF, phenylmethanesulfonyl fluoride; CMC, critical micelle concentration; CD, circular dichroism; NMR, nuclear magnetic resonance
The conformational conversion of the cellular prion protein (PrPC) to the β-rich infectious isoform PrPSc is considered a critical and central feature in prion pathology. Although PrPSc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrPC to proteinase K resistant PrPSc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrPC conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrPC to PrPSc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).
Introduction Prion diseases or transmissible spongiform encephalopathies (TSEs), including Kuru, variant Creutzfeldt Jakob disease (vCJD), bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), and scrapie are all examples of incurable and uniformly fatal neuro-degenerative diseases that arise from the self-propagation of misfolded prion proteins. The current working hypothesis is that small numbers of misfolded, β-rich prions (called PrPSc) are able to catalytically convert native, monomeric, helix-rich cellular prion protein (PrPC ) to large numbers of misfolded prions,1 which aggregate into larger, uniformly organized, oligomeric substructures that can further assemble into fibrillar, macrostructures. In this way, the misfolded prions are able to spread exponentially leading to loss of neuronal function wherever PrPC is highly expressed. PrPC is found in all mammalian cells; but the highest level of expression is in neurological tissues,2,3 which largely explain the neurodegenerative nature of prion diseases.
The native prion protein, PrPC, is a lipid-bound, monomeric, ~24 kDa protein with an unstructured N-terminal region containing roughly 70 residues and a globular core consisting primarily of α-helical secondary structure4 (42% α-helix and about 5% β-sheet). This unstructured N-terminus consists of 5 octa-repeat segments that have been shown to bind divalent metal ions (Cu2+, Fe2+, Zn2+).5,6 The core contains 2 N-linked glycosylation sites at Asn181 and Asn1977,8 while the C-terminus is linked to a lipid glycosylphosphatidylinositol (GPI) anchor that secures the protein to the cellular membrane. PrPSc is rich in β-sheet structure that contains 10% α-helix and 30% β-sheet.1,9,10 However, a recent study showed that PrPSc consists largely of β-strands with no α-helices.11 These β-sheets tends to form large oligomers, insoluble aggregates, and fibril like structures. As a result, PrPSc is protease-resistant, oligomeric, and substantially less soluble than PrPC. These divergent features allow the 2 isoforms to be easily differentiated biochemically. After many decades of investigation, much is known of the prion protein’s (PrPC) native structure; however, less is known about the
*Correspondence to: David S Wishart; Email: [email protected] Submitted: 01/01/2014; Revised: 04/10/2014; Accepted: 04/16/2014; Published Online: 05/12/2014 http://dx.doi.org/10.4161/pri.28939 www.landesbioscience.com Prion 221
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Keywords: prion protein, protein misfolding, lipopolysaccharide, beta oligomer, fibril
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continued exposure to detergents. Despite the widespread belief in the “protein-only” hypothesis concerning prion protein conversion and infectivity, controversies over whether other molecules beside PrPSc are also required for conversion and replication in vivo, still remain. In particular, the role of a number of naturally occurring, non-proteinaceous molecules such as nucleic acids,19 polyanions,15,20 lipids,21-23 sulfated glycans,15 and metals24 are actively being investigated in the pathogenesis of prion diseases. Recently, it was shown that recombinant prion proteins could be rendered seed-competent (and infectious) with the addition of total liver RNA and synthetic 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) lipid molecules.20 Even more recently it was reported that phosphatidylethanolamine (PE) is an essential cofactor for generating infectivity from converted recombinant PrP.25 These findings strongly suggest that a non-protein molecular component may be responsible for facilitating or augmenting the prion protein conversion process.19,25 Given these emerging results, we began screening for a “prion protein converting molecule” among naturally occurring, exogenous molecules. The recent discovery that the N-terminal residues of the human prion protein (PrPC ) has broad spectrum antibacterial properties26 mediated by membrane-prion protein interactions directed our screen toward analyzing bacterial components and specifically looking at lipopolysaccharide (LPS), an outer membrane component of gram-negative bacteria. In particular, LPS is a temperature resistant, amphipathic molecule that consists of both lipid (lipid A) and carbohydrate (core and O-antigen) components. LPS acts as an immunogen and a strong pyrogen in healthy mammals. It binds the CD14/ Figure 1. Region of the 15N-HSQC NMR spectra for the ShPrP90–232 TLR4/MD2 receptor complex which resides in lipid rafts before (A) and after (B) the addition of an equimolar amount of LPS. where the prion protein co-localizes.27 LPS promotes the secretion of pro-inflammatory cytokines in many cell β rich isoform. Likewise, very little is known about the α-helix to types, but especially in macrophages. Given that both β-sheet conversion process, the precise mechanisms behind self- PrPC and the LPS/CD14/TLR4 receptor complex are localized seeded propagation or the potential causative agents that might to the same membrane domains via their GPI anchors and given induce sporadic prion disease. As prion protein misfolding and the structural similarity of LPS to polyanionic molecules like aggregation is believed to be a major contributor to prion disease POPG that have been associated with prion protein conversion, etiology, a more detailed molecular understanding of the ordered we decided to investigate LPS’s potential effects on ShPrP (90– aggregation and amyloid fibril formation process is critical for 232) in vitro. developing strategies to prevent or treat these conditions. Interestingly, we found that native, recombinant prion To help better understand biochemical aspects of PrP protein incubated with modest concentrations of LPS (15 μg/ conversion, several groups have used in vitro or test-tube systems mL) at physiological normal pH and temperature led to the in which recombinant PrPC molecules are converted into to β-rich rapid conversion to oligomers that were PK resistant and rich in PrPSc-like molecules (PrPβ) using a number of chemical and/or β-sheet structure (PrPβ) compared with the native ShPrP (90– physical agents such as guanidine hydrochloride,12 urea,13 SDS,14 232). Furthermore this conformational conversion to a β-sheet high temperature,15 low pH16 that cause complete or partial rich structure occurs at ratios of PrP to LPS below 1:1 (w/w), as protein denaturation. Most of these protocols not only cause long as the LPS concentration in the conversion solution remains the conformational change of PrPC into PrPβ but also generate above the critical micelle concentration (CMC). Even more “synthetic prions” that exhibit many of the physiochemical interestingly, this conversion phenomenon, whereby conversion to properties seen in PrPSc molecules isolated from diseased tissues.17,18 a β-sheet rich structure occurred down to a weight ratio of ShPrP However, unlike brain-derived prions, the conversion and to LPS of 1:0.09 (w/w), was not seen with other known prion apparent propagation of these synthetic prions is dependent on conversion reagents (including SDS, urea, POPG, or PE). Given
the ubiquity of LPS in both recombinant prion preparations and the abundance of LPS in many animal pens, cages, and feed, these results may have some interesting implications regarding prion conversion – both in vitro and in vivo.
Results LPS mediated conversion The strong interaction between the prion protein and bacterial LPS was evident from our initial binding studies performed by NMR (Fig. 1). Apart from a few N-terminal and 6x-His affinity tag residues, immediate and complete attenuation of ShPrP (90–232) 15N-HSQC amide signals resulted upon addition of a 1:1 ratio (w/w) of LPS to the ShPrP (90–232) sample. This interaction was also visualized by transmission electron microscopy (Fig. 2). As seen in Figure 2, the typical cylindrical bicellular structure adopted by LPS in water was disrupted leading to a spherical micelle formation (Fig. 2B) with micelle diameters ranging between 50 and 150 nanometers. The negative staining achieved with uranyl acetate suggests that protein is adhering to the micelle surface. More interestingly, conversion to a fibril state was observed upon incubation of this complex at 37 °C (Fig. 2C and D). These converted prion protein fibrils ranged in length from 100 nm to 1 μm and were associated in pairs and clustered with the LPS micelles. To further characterize this state, a circular dichroism
(CD) spectrum was collected (Fig. 3B) and a proteinase K (PK) digestion assay was performed (Fig. 3A). The CD spectrum revealed a high level of β-sheet structure and the PK digestion revealed a 12 kDa proteolytic resistant core, consistent with both β-oligomeric28 and spontaneous PMCA-derived isoforms.29 To test if this phenomenon was restricted to Syrian hamster prion protein we also found that mixing pure LPS in the presence of NaCl (150 mM) with recombinant mouse prion protein at mg ratios (w/w) of 1:1 (LPS:ShPrP (90–232) also resulted in instant conversion to PrPβ (result not shown). These preliminary findings warranted a more in-depth characterization of the LPS mediated conversion process. In particular we decided to investigate the effect of differing LPS concentrations on the prion conversion process. Figure 4 shows the results of the LPS-mediated conversion in which solutions with progressively lower ShPrP (90–232) to LPS ratios were prepared and monitored by CD. Weight (w/w) ratios above 1:0.5 (ShPrP (90–232):LPS) resulted in the complete conformational change of PrPC into β-sheet oligomers. Meanwhile, solutions in which the LPS was below the critical micelle concentration (CMC) of 14 μg/mL did not show any conversion at all, even after 4 wk of incubation. Thus we conclude that a micellar form of LPS (as might be found in a live or lysed bacterial cell) is required for initiating conversion. The secondary structure content (% α-helix and β-sheet) of the prion proteins were calculated from the CD data presented in Figure 4 for each dilution and are shown in Table 1.
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Figure 2. Electron microscope images of the LPS mediated conversion of the ShPrP (90–232) protein. (A) LPS micelles at 0.5 mg/mL in water, pH 7.0 (magnification 71 000×, scale bar 50 nm). (B) The same LPS “micelles” shown in (A) after addition of 0.5mg/mL ShPrP (magnification 56 000×, scale bar 200 nm). (C and D) The sample after the addition of 150 mM NaCl, 0.1% NaN3 and incubated at 37 °C for 48 h magnification 110 000× and 14 000×, scale bars: 50 nm and 0.5 um respectively). Inset in Figure 2D was collected at 110 000× (scale bar 200 nm). Small fibrils can be seen in C. Samples were stained with lead acetate (A) or uranyl acetate (B–D).
(with PrPβ) was serially diluted by half, by adding equal volumes of fresh recombinant ShPrP (90–232) of the same concentration to the LPS converted PrPβ. This serial dilution process generated subsequent solutions with ShPrP (90–232) to LPS ratios (w/w) of 1:6, 1:3, 1:1.5, 1:0.75, 1:0.38, 1:0.17, 1:0.09, and 1:0.01 (Fig. 4; Table 1). Interestingly conversion of ShPrP (90–232) to a β-sheet rich structure (above 20% β-sheet) was maintained even when the molar ratio (mol/mol) of LPS to ShPrP (90–232) fell below 1:1 (equivalent to 1:0.38 w/w ratio of ShPrP (90–232):LPS). This is unlike other conversion methods that only showed a dilution effect upon addition of fresh ShPrP (90–232). This confirmed that a direct interaction and conversion was being observed between LPS and the protein. It also confirmed that the measured conformational change was not simply due to a denaturation effect. This conversion was observable at all LPS concentrations, until the LPS concentration dropped below its CMC (ShPrP (90– 232):LPS ratio (w/w) of 1:0.01). This effect is likely due Figure 4. CD spectra for 25 μM of ShPrP (90–232) with various concentration (w/w to the sensitivity of the experimental method used to ratios) of LPS (1:6 300 μM LPS, 1: 3–150 μM LPS, 1:1.5–75 μM LPS, 1:0.75–37.5 μM detect the change. More importantly, this serial dilution LPS, 1:0.38–18.5 μM LPS, 1:0.17–9.5 μM LPS, 1: 0.09–5 μM LPS, and 1:0.01–1 μM effect was used with RENAGE gel analysis (discussed LPS). Spectra were collected immediately after adding LPS, except for the 1:0.01 below) to ascertain the molecular interaction ratio. w/w ratio of LPS with shPrP(90–232), which was incubated an additional 4 wk to ascertain any changes in secondary structure. LPS-mediated PrPβ conversion and its subsequent serial dilution was compared with the conversion and Serial dilution serial dilution mediated by other chemical denaturants: Solutions containing LPS, unconverted recombinant ShPrP namely SDS, POPG, DOPE, and urea (Fig. 5; Table 2). While (90–232), and LPS-converted PrPβ (in various concentrations) LPS, SDS, POPG, and PE all share lipid moieties, only PE has a was also used to assess the unique concentration dependence positive charge or a cationic group provided by a primary amine. property of LPS in PrP conversion. Mixing pure LPS in the Furthermore, while SDS and POPG both contain negatively presence of NaCl (150 mM) with recombinant ShPrP (90– charged polar heads only LPS contains a phosphate group along 232) at a ratio (w/w) of 1:6 (ShPrP (90–232):LPS) resulted in with a complex saccharide. LPS-generated PrPβ demonstrates a instantaneous conversion to a PrPβ isoform (measured by the capacity to convert prion protein even at sub molar ratios as the percentage of helical and β-sheet structure determined by CD). concentration of LPS is reduced (down to its CMC) in contrast After confirming the conversion, the LPS-containing solution to all other reagents which show a progressive dilution effect. In
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Figure 3. (A) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrPβ . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the right). Lane 5: PK:ShPrPβ 1:1000. Lane 6: PK:ShPrPβ 1:750. Lane 7: PK:ShPrPβ 1:500. Lane 8: PK:ShPrPβ 1:250. (B) CD spectra for the ShPrP (90–232) with 1:1 w/w ratio of LPS at 0 incubation time.
Table 1. Secondary structure content (measured by CD) of LPS-converted prions α-Helix (%)
β-Sheet (%)
42
9
9
32
ShPrP (90–232): LPS (1:3)
10
31
ShPrP (90–232): LPS (1:1.5)
8
33
ShPrP (90–232): LPS (1:0.75)
12
31
ShPrP (90–232): LPS (1:0.38)
17
29
ShPrP (90–232): LPS (1:0.17)
20
24
ShPrP (90–232): LPS (1:0.09)
26
20
ShPrP (90–232): LPS (1:0.01)
41
10
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ShPrP (90–232) control ShPrP (90–232): LPS (1:6)
Figure 5. CD spectra for the ShPrP (90–232) with various molar ratios of DOPE, SDS, POPG, and urea at 0 incubation time. (A) CD spectra of the prion propagation studies using DOPE (diluted from 1:64 to 1:16 PrPC:DOPE (w/w)). (B) Urea induced oligomers and fibrils (for comparison of secondary structure content). (C) SDS (1:0.2 to 1:0.0125 PrPC:SDS). D. POPG (diluted from 1:0.5 to 1:0.065 of ShPrP [90–232]:POPG). Each diluted sample was incubated an additional 19 d to ascertain any changes in secondary structure.
other words, as the concentration or urea, POPG, and SDS is reduced, the extent of conversion is reduced. Interestingly SDS can only convert ShPrP (90–232) to PrPβ at concentrations
below its CMC of 8 mM (0.23% w/v). On the other hand conversion with urea and salt is time dependent with a faster conversion below the denaturation midpoint of 3.6 M.30 This
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Sample (condition)
Table 2. Secondary structure content of urea, DOPE, POPG, and SDS mediated conversion of prion protein DOPE induced conversion
*Urea induced conversion
Sample
α-Helix (%)
β-Sheet (%)
Sample
α-Helix (%)
β-Sheet (%)
ShPrP: DOPE (1:64)
41
10
ShPrP Native
43
11
ShPrP: DOPE (1:32)
40
12
Urea-induced ShPrP Oligomers
14
23
ShPrP: DOPE (1:16)
40
13
Urea-induced ShPrP Fibrils
10
27
POPG induced conversion
Sample
α-Helix (%)
β-Sheet (%)
Sample
α-Helix (%)
β-Sheet (%)
ShPrP: SDS (1:0.2)
13
31
ShPrP: POPG (1:0.5)
12
30
ShPrP: SDS (1:0.1)
27
18
ShPrP: POPG (1:0.25)
35
14
ShPrP: SDS (1:0.05)
38
13
ShPrP: POPG (1:0.125)
38
12
ShPrP: SDS (1:0.025)
40
10
ShPrP: POPG (1:0.0625)
42
8
ShPrP: SDS (1:0.0125)
42
8
Each diluted sample was incubated an additional 19 d to ascertain any changes in secondary structure. *Urea induced conversion: secondary structure values calculated for urea/guanidine converted β oligomers and fibrils used to compare with secondary structure values of controls (DOPE,SDS, and POPG) and LPS converted ShPrP.
suggests the importance of a partially unfolded intermediate in urea induced conversion. POPG has been shown to facilitate the conversion of recombinant PrP into an infectious strain capable of propagating in mice.20 POPG contains 2 alkyl carbon chains attached to a dihydroxylated phosphate group. Like LPS, it requires concentrations above its CMC (and therefore in lipid vesicles) to convert recombinant ShPrP (90–232) to PrPβ.31 However, unlike LPS, the extent of POPG conversion appears to be directly proportional to the concentration of POPG used (Fig. 5D), as the concentration of POPG is reduced, the extent of conversion is largely reduced. Quite recently it was also reported that phosphatidylethanolamine (PE or DOPE) can act as a solitary cofactor in the propagation of recombinant PrPSc using brain homogenate and standard PMCA conditions.25,32 To explore this further, we tested whether the same concentrations of DOPE described in the original publication25 could facilitate the conversion of ShPrP (90–232) to a β-sheet conformation without brain homogenate and sonication. Interestingly, our results showed that no conversion to PrPβ occurred, even after 7 d of incubation at 37 °C (Fig. 5A). These findings suggest that other factors, such as temperature or additional brain homogenate cofactors are responsible for initiating the conversion process mediated by PE. Given that the previous propagation studies of both DOPE and POPG were used in conjunction with other cellular components (cell extracts, RNA), it suggests that additional agents are required to mediate the conversion of PrPC to a propagating isoform. Our findings suggest that saccharide moieties (which are found only in LPS) may have the necessary requirements to facilitate conformational conversion. Size comparison of PrPβ formed by conversion and fibril propagation To compare the size of PrPβ aggregates formed by the various prion protein conversion techniques we used a native gel electrophoresis technique called RENAGE.33 With RENAGE it has been previously shown that the PrPβ oligomers formed via
urea/salt conversion consist of a distribution of heptamers to dodecamers.33 This size distribution of medium-sized oligomers is seen in Figure 6. In contrast, LPS-converted prion protein (PrPβ) are characterized by very large aggregates that migrate just a few millimeters into the stacking gel. The large size of these oligomers may be due to the presence of tightly bound LPS. Quantifying the protein bands for the LPS-converted prion proteins at 1:0.09 PrP:LPS shows that 19% of PrP is found in the large aggregate band, 15% is found in oligomers, 6% is in the dimer band and the rest is in the monomer band. Given that the molecular weight of monomeric LPS is roughly half that of the prion protein, the 1:0.09 weight (w/w) ratio equates to a 1:0.18 molar (mol/mol) ratio. This correlates strongly with the 19% protein band shown in RENAGE. An equimolar component of the protein is found in the oligomers. We propose that these oligomers are loosely associated with the tightly bound protein. More importantly, this loosely bound protein, while dissociable from the micellar bound component, still persists in a β-sheet rich, oligomeric state. Based on a two-state model, the native α-helical prion protein (ShPrP (90–232) has an α helix content of 42% and β-sheet content 9% while the LPSconverted PrPβ has only 10% α helix and 32% β-sheet structure (Table 1). If only the oligomeric, LPS-bound 18% of protein was in fact converted, the resulting CD spectra of the 2 states should present CD spectra showing 36% helix and 14% β sheet. Instead, a calculated CD spectra for 40% converted PrPβ protein and 60% unconverted (ShPrP [90–232]) monomer would result in a CD spectra with 29% α helix and 18% β sheet composition, which is close to the observed values (Table 1). Thus, twice the amount of β-sheet structure in LPS-converted PrPβ than predicted for a 1:0.18 ShPrP (90–232):LPS molar ratio, indicates a stoichiometric interaction ratio of 2:1 ShPrP (90–232):LPS. RENAGE was also used to compare oligomer sizes and structure induced by POPG and SDS. As seen in Figure 6, RENAGE of the POPG-converted prion protein (PrPβ ) shows only a small amount of aggregation. The POPG-converted oligomers appear
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SDS induced conversion
Figure 6. Resolution enhanced native acidic gel electrophoresis (RENAGE) shows different size and stability of oligomers formed from the different conversion methods. Conversion of recombinant ShPrP (90–232) is compared for urea/salt conversion (3M urea 20 mM sodium acetate pH 4 and 200 mM NaCl), POPG conversion (0.05:1 POPG to PrP [w/w], incubated at 37 °C for 4 d), SDS conversion (0.02% SDS), and LPS conversion (1:0.09 PrP to LPS, incubated at 37 °C for 2 d), 8 μg of each sample is loaded. The latter is from MoPrP (90–231) cross-linked nonspecifically using PICUP.
initiating the PrP conversion reactions and that nucleic acids do not mediate the conversion or influence the LPS-directed conversion.
Discussion In this study we have shown that LPS interacts with recombinant prion protein and that this interaction converts the α-helix-rich recombinant ShPrP (90–232) into a β-rich, PK-resistant form (PrPβ) under physiological conditions. LPS induced PrPβ conversion does not require any brainderived PrPβ seed, additional co-factors, or denaturants. This is in marked contrast to most other conversion and propagation protocols.14,16,20,29,38,39 Furthermore, many published conformational prion protein conversion strategies rely on a molar excess or destabilizing amounts of chemical denaturants (urea, guanidine hydrochloride), synthetic detergents (SDS), or extremes of pH or high (denaturing) temperatures.12-16 However, LPS-mediated prion protein conversion is distinct from these methods in that measurable conversion to a β-sheet rich form occurs at ShPrP (90–232) to LPS ratios down to 1:0.09 (w/w). Additionally, sizing of oligomers using RENAGE shows that the LPS converted PrPβ oligomers differ from those generated by POPG, SDS, and urea. Furthermore, RENAGE data showed that the LPS-generated PrPβ oligomers will dissociate to monomers. This differs from PrPβ oligomers formed by urea (Fig. 6) or acid,33 which are stable under electrophoresis conditions.
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to be unstable under native electrophoresis. Similarly, conversion of ShPrP (90–232) with 0.02% w/v SDS resulted in large aggregates that appear to dissociate as electrophoresis proceeds. While both the SDS and POPG conversion techniques produced β-rich oligomers with about 30% β sheet content (Fig. 6), only the POPG formed oligomers could dissociate into a monomeric state, while the SDS formed PrPβ remained in a loosely associated oligomeric state. Efforts to generate PrPβ oligomers or fibrils using DOPE were not successful and so they were not studied by RENAGE. In contrast, the LPS generated PrPβ is distributed into various states (Fig. 6). We also found that mixing LPS with recombinant cervid prion protein or recombinant mouse prion protein at a weight ratio of 1:1 LPS:ShPrP (90–232) (w/w) also resulted in conversion to large aggregates (result not shown). Nucleic acid control experiments Discrepancies exist in the literature regarding the necessary components required to induce conversion of the prion protein into a self-replicating form.15,20,23,34,35 While cofactors are not necessary for converting recPrPC to PrPSc in prion seeded PMCA,36 cellular components like RNA and lipids are required to generate de novo, self-propagating prions from recombinant prion protein sources.19,20 This issue needed to be addressed given that the commercial sources of LPS are contaminated with various amounts of nucleic acids (RNA, DNA, short oligos, and free NTPs). To rule out the potential effects that certain bacterial cellular components from E. coli might have on our observed reactions we sought to control for these known LPS contaminants. While RNA is reported to interact with N-terminal residues of PrP,35,37 DNA has been shown to interact with the C-terminal residues of the prion protein.34 Given that our recombinant prion protein construct is devoid of any reported RNA binding site and that our lab does not control for RNase activity, the influence that RNA might have on the observed conversion was of less concern than that of DNA. Two sources of LPS were obtained from Sigma. One preparation (L3012) is purified by gel filtration and is reported to have >10% nucleic acid contamination, while the other (L3024) is further purified by ion-exchange chromatography and contains
