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1 Department of Poultry Science and 2 Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA
Reprint requests to: Ching-Ying Chen, Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7608, USA; e-mail: cchen8{at}ncsu.edu; fax: (919) 515-2625.
(RECEIVED November 11, 2004; FINAL REVISION May 24, 2005; ACCEPTED May 31, 2005)
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Keywords: BSE; prion; PrPSc; Sup35NM; yeast prion; prion surrogate protein; enzymatic degradation
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041234405.
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Introduction |
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The pathological isoform of prion protein (PrPSc) aggregates in diseased tissues and, in common conditions, is resistant to digestion by proteases (Caughey et al. 1991; Pan et al. 1993). It was recently found that PrPSc in diseased tissues can be degraded by a feather-degrading keratinase and other microbial proteases when the tissue homogenate was preheated at 115°C (Langeveld et al. 2003). Independently, mouse bioassays showed a significant reduction of infectivity after a proteolytic inactivation process (McLeod et al. 2004). These results indicated a potential method of enzymatic inactivation of prion protein.
PrPSc is pathogenic and dangerous to handle. A mutant recombinant prion produced in Escherichia coli without glycosylation was recently found to be able to induce neurologic dysfunction (Legname et al. 2004). Experimentation with prion tissues must be strictly controlled and is inordinately expensive. A nonpathogenic prion surrogate protein which has similar physical-chemical properties will be useful for research in this field. In addition, it may be used as a marker to test or screen putative prion inactivation methods.
The yeast with prion-like proteins was once thought to be useful as an in vivo model to screen for anti-prion drugs (Saupe 2003). Sup35p from Saccharomyces cerevisiae is a translation termination factor that can convert into an insoluble aggregate, a property similar to prion protein (Masison and Wickner 1995; Paushkin et al. 1996). The structure of Sup35p can be divided into three regions, namely, N, M, and C, based on their positions and different functions. Individual regions and combinations have been cloned, expressed, and purified from E. coli. They were characterized for their properties to form aggregates, or amyloid, in vitro (Derkatch et al. 1997; Glover et al. 1997; King et al. 1997; Osherovich et al. 2004). The aggregates of Sup35N and Sup35NM were found to be proteinase K-resistant (Kushnirov et al. 2000).
For these reasons, Sup35NM was selected for the present study of production, purification, and characterization. Its degradation by keratinase and proteinase K under various conditions was also studied and compared. Being nonpathogenic, Sup35NM could be a candidate for a safe prion surrogate protein.
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). After heating to 100°C for 10 min, the fibrils disappeared (Fig. 1B).
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). However, SDS-PAGE and Western blotting analyses showed that the samples were not fully aggregated, with soluble monomeric Sup35NM still detectable after 5 h. At 22–48 h, soluble protein was almost gone. Heating the same 48-h sample at 100°C for 10 min produced a band with the same mobility and intensity as those of the band at 0-time (Fig. 2B,C). Insoluble aggregates that stuck in the loading wells of the gel did not stain well by Coomassie blue (Fig. 2B), but they could be transferred onto PVDF membrane by Western blotting and detected by immunostaining (Fig. 2C). This result also indicated that Congo red binding is not a good indicator for the aggregation of Sup35NM. Western blotting clearly showed a gradual increase of amyloid Sup35NM as the insoluble protein trapped in the loading wells. After heating the 48-h sample at 100°C for 10 min, insoluble amyloid disappeared and the soluble Sup35NM reappeared (Fig. 2B,C). As observed with electron microscopy (Fig. 1B
), deaggregation of fibrillar Sup35NM resulted from heating at 100°C for 10 min.
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). However, the binding of Thioflavin T (ThT), as measured by fluorescence emission, clearly showed that unseeded Sup35NM took 24–48 h of slow rotation to complete aggregation (Fig. 3A). Samples at each time point also were analyzed by SDS-PAGE (Fig. 3B). Along with aggregation time, fluorescence emission increased and monomeric Sup35NM in SDS-PAGE decreased. The comb of loading wells is not shown in Figure 3B, as in Figure 2B. The insoluble fibrils in the wells were not stained by Coomassie blue.
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). SDS-PAGE analysis indicated complete degradation at the high enzyme level (7.3 U) and incomplete degradation at lower enzyme levels, where two different enzymes produced different patterns of protein fragments (Fig. 4A,D). Western blotting probed with anti-M serum (Fig. 4B,E) and anti-N serum (Fig. 4C,F) clearly showed that the high enzyme level (7.3 U) resulted in more degradation.
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). For 37°C digestion (Fig. 5A,C,E), preheating at 115° and 100°C made the amyloid degradable. After preheating at 80°C, or no preheating, the amyloid was resistant to the digestion when the enzyme level was 1.5 U and became less resistant when the enzyme level was increased to 7.3 U (Fig. 5A,C,E). For 50°C digestion, after preheating at 80°C or no preheating, again the amyloid was resistant to digestion at the low enzyme level (1.5 U). However, at the high enzyme level (7.3 U), the amyloid, regardless of whether the sample was preheated, was degradable (Fig. 5B,D,F).
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). Digestion at the high enzyme level produced the same result. Preheating rendered Sup35NM amyloid readily degradable (Fig. 6B,D,F), the same as was found for the BSE prion protein (Langeveld et al. 2003).
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), and the different conditions for enzymatic degradation were tested.
The process of Sup35NM aggregation was analyzed by Congo red binding, fluorescence of ThT binding, SDS-PAGE, and Western blots. On a time course from 0 to 48 h, samples of aggregating Sup35NM were analyzed by SDS-PAGE. At the same time points, Congo red and ThT samples were also prepared and analyzed. A gradual increase in Congo red binding was found from 0 to 5 h before leveling off (Fig. 2). A gradual increase in ThT binding was detected during the course of 24 h before total saturation (Fig. 3). The agreement of increase of ThT fluorescence with the decrease of monomeric Sup35NM was confirmed by SDS-PAGE analysis (Fig. 3). The difference between the two spectrometric methods is believed to be due to the fact that Congo red binding detected changes in protein folding, specifically, the formation of 
-sheets or the early stages of -oligomer formation, as opposed to the larger aggregates of Sup35NM that ThT fluorescence detects (Baskakov et al. 2002). For this reason, ThT binding monitored by fluorescence emission should be the method of choice for monitoring the formation of amyloid fibrils.
In our previous study, PWD-1 keratinase and some other proteases were found to be able to degrade PrPSc in brain stem tissues from infected cattle and sheep (Langeveld et al. 2003). However, preheating treatment of the tissue homogenate is needed for complete digestion of PrPSc by these enzymes. It is possible that the preheating at 115°C caused a change in the structure of the PrPSc subjected to enzymatic degradation. In the present study with Sup35NM aggregates or fibrils, the same scenario was observed. Compared with its monomeric form, amyloid Sup35NM was found to be relatively resistant to both keratinase and proteinase K (Fig. 4).
The keratinase may be slightly more active than proteinase K in degrading polymeric Sup35NM when they were at the same level of enzyme units. More studies will be needed to determine the difference in activity of these two enzymes against prions. More importantly, both enzymes produced defined patterns of degradation products or fragments, and they are different (Fig. 4). This indicated that the cleavage sites of keratinase and proteinase K are specific and different. It will be of interest to identify these enzyme-specific cleavage sequences. The incomplete digestion can provide a characteristic marker of Sup35NM as the surrogate protein of prions.
In conclusion, various conditions to optimize partial and complete degradation of Sup35NM amyloid fibrils were studied (Figs. 5, 6). Ultimately, the most effective means will be verified with infected brain tissue and PrPSc and determined by infectivity tests in animals. Sup35NM aggregates are less resistant to enzymatic degradation than PrPSc in BSE tissue, indicating the difference in structures. To fulfill the role of a safe, simple, and cost-effective surrogate protein for screening and optimizing experimental conditions, more comparative studies between Sup35NM and PrP are needed.
Since the discovery of enzymatic degradation of prion protein (Langeveld et al. 2003; McLeod et al. 2004), it will be of great interest to learn more about the interaction of the enzyme and prion protein and the mechanism of degradation. Further studies will focus on the protein intermediates produced from partial degradation to determine the cleavage sites in the Sup35NM amyloid structure. Also, it is possible that deaggregation and unfolding of the polymeric structure are the prerequisite steps, such as by preheating, for the proteolytic hydrolysis that follows. Being nonpathogenic and fairly easy to prepare, Sup35NM is a good candidate as a prion model for many research and technological applications. A similar protein molecule was recently developed and tested for its prion-like properties (Wang et al. 2005). Sup35NM and this molecule will be compared to determine the abilities of each to serve as a safe prion marker for disinfection processes and metabolic studies. In this study, we specifically evaluated Sup35NM properties.
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Materials and methods |
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-D-thiogalatoside (IPTG), sodium phosphate, potassium phosphate, azocasein, Congo red, and proteinase K were from Sigma Chemicals. Urea was from Promega. Thioflavin T was from Acros Organics. The Q Sepharose fast flow and the ECL Western blotting analysis system were from Amersham Pharmacia. The CHT ceramic hydroxyapatite, the PVDF membranes, and the anti-rabbit antibody conjugated to horseradish peroxidase were from Bio-Rad. The chemiluminescence reagent was from Pierce. Polyclonal antibodies produced by Biosource based on the epitopic peptide sequences, Ac-QGGYQQYNPDAGYQ-amide (Liu et al. 2002) and Ac- CAPKPKKTLKLVSSSG-amide (T.R. Serio, pers. comm.), were used to generate anti-Sup35NM polyclonal antisera against amino acids 55–68 (anti-N) and 135–148 (anti-M), respectively. Keratinase was produced and purified as described (Lin et al. 1992, 1997).
Preparation of Sup35NM
The expression plasmid pJC25NM stop was a gift from Dr. Susan L. Lindquist (Whitehead Institute, Massachusetts Institute of Technology). Sup35NM was purified as described (Chernoff et al. 2002) with the following modification. Transformed colonies were inoculated into a 50-mL LB-ampicillin (50 µg/mL) flask and incubated for 5 h at 37°C, and then 20 mL of the growing culture was removed and added into a fresh 500-mL LB-Amp (50 µg/mL) medium. After induction with 1 mM IPTG at an A600 of ~0.6, cells were harvested after 3 h of induction. Cell pellets were collected by centrifugation and stored at –80°C. Cells were lysed in 50 mL lysis buffer (10 mM Tris-HCl [pH 7.2], 1 mM DTT, 1 mM PMSF, 8 M urea) for 30 min at 25°C. The lysate was then cleared by centrifugation at 12,000g for 20 min at 10°C. The cleared supernatant was applied to a Q Sepharose column (2.5x7.5 cm) pre-equilibrated with lysis buffer. The column was washed with 5x bed volumes of Q wash buffer I (10 mM Tris-HCl [pH 7.2], 1 mM DTT, 1 mM PMSF, 8 M urea, 85 mM NaCl), and 5x volumes of Q wash buffer II (10 mM Tris-HCl [pH 7.2], 8 M urea, 150 mM NaCl). The protein was eluted in 3 volumes of Q elution buffer (10 mM Tris-HCl [pH 7.2], 8 M urea, 200 mM NaCl). The eluant from the Q Sepharose was then loaded directly onto a hydroxyapatite column (1.5x12 cm). The column was pre-equilibrated with Q elution buffer. After loading, the column was washed with 2x volumes of HA wash buffer I (1 mM potassium phosphate [pH 6.8], 8 M urea, 1 M NaCl) and then with 2x volumes of HA wash buffer II (25 mM potassium phosphate [pH 6.8], 8 M urea). The protein was eluted using a stepwise gradient of 75 mM and 125 mM potassium phosphate (pH 6.8) in 8 M urea and then with 2x volumes of 125 mM potassium phosphate (pH 6.8) in 8 M urea. Fractions (5 mL) were analyzed by 15% SDS-PAGE (loading 10 µL per lane). Protein concentrations were determined with the calculated extinction coefficient of 0.90 for a 1 mg/mL Sup35NM solution at 280 nm absorbance (Gill and von Hippel 1989). Anhydrous methanol (100%) was added to precipitate Sup35NM on ice at a ratio of 5:1. The supernatant was removed and the pellet was stored in 70% (v/v) methanol (1/2 volume of supernatant) at –80°C (Serio et al. 1999).
Aggregation and deaggregation of Sup35NM
Methanol-precipitated Sup35NM was resuspended in 25 mM potassium phosphate (pH 8.0). Sup35NM after sitting at 25°C for 3 d can aggregate into fibrils. These fibrils can be used as seeding material. Typically, 2% preformed fibrils were added to freshly suspended Sup35NM to accelerate aggregation. Alternatively, the resuspended Sup35NM in buffer was allowed to undergo a slow rotation at 5 rpm. Both methods were carried out at room temperature. For deaggregation, amyloid Sup35NM was boiled at 100°C for 10 min.
Electron microscopy
Sup35NM protein was negatively stained according to Chernoff et al. (2002). Here, 5 µL of a 5 µM protein solution was applied to a glow-discharged 400 mesh carbon-coated copper grid followed by staining with several drops of 2% (w/v) aqueous uranyl acetate, then air-dried. Samples were then observed in a JEOL–100S Transmission Electron Microscope at an accelerating voltage of 120 kV.
Congo red binding assay
Congo red binding assays were carried out as described previously (Klunk et al. 1989). Congo red was dissolved in Congo red binding buffer (CRBB) (5 mM potassium phosphate [pH 7.4], 150 mM NaCl) to give a concentration of 10 mM. Methanol-precipitated Sup35NM was resuspended in CRBB to 10 µM. Samples were first heated at 100°C for 10 min to deaggregate the preformed fibrils, and reaggregation was started by incubation at 25°C. At indicated times, protein was diluted to 2 µM with 10 µM Congo red in CRBB and incubated for 30 min at 25°C before measuring. To calculate moles of Congo red bound/L of solution, the following equation was used: mol CRbound/L=(A540/25295) – (A477/46306).
Thioflavin T binding assay
The Thioflavin T (ThT) assay was carried out as described (Chernoff et al. 2002). A stock solution of 1 mM ThT in water was prepared fresh for each experiment. Methanol-precipitated Sup35NM was resuspended in CRBB to 10 µM. Samples were heated at 100°C for 10 min and then started to aggregate at 25°C as described above. At indicated times, Sup35NM protein was diluted to 0.2 µM in the presence of 20 µMThT in CRBB. ThT fluorescence was monitored using a Quantech fluorometer (Barnstead Thermolyne), with excitation at 450 nm and emission at 481 nm (excitation slit, 1 nm; emission slit, 10 nm; 0.4-cm cuvette).
Azocasein assay
The keratinase and proteinase K activities were measured as described (Lin et al. 1992, 1997) with some modification. For a standard assay, 5 mg of azocasein was added with 0.8 mL of 50 mM potassium phosphate buffer (pH 7.5). Completely suspended azocasein solution was preheated at 37°C for 10 min. Five µL of appropriately diluted enzyme solution was added, and the mixture was incubated at 37°C for 10 min. For the control reaction, 5 µL of diluted buffer was added to azocasein solution. The reaction was terminated by the addition of 0.2 mL of 10% trichloroacetic acid (TCA) and put on ice for 10 min. Samples were centrifuged (14,000g, 4°C, 10 min), and the released azo-peptides were in the supernatant. The absorbance of the supernatant was measured with a spectrophotometer (Shimadzu Scientific Instruments). In this assay, we defined 1 U of enzyme activity as an increase in A450 of 0.01 after 10 min in reaction compared to the control.
Protease resistance of amyloid Sup35NM
This method was modified from a Sup35pN proteinase K resistance assay (King et al. 1997). About 50 µg of Sup35NM samples, monomeric or amyloid Sup35NM, were suspended in 25 µL of 25 mM potassium phosphate buffer (pH 8.0). Enzyme (2 µL) was added and the mixture was incubated for various periods of time at the regular assay temperature, 37°C, or at the keratinase optimum temperature, 50°C (Lin et al. 1992). Reactions were terminated by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM. SDS sample buffer was added at 1x and boiled for 10 min. Samples were run on a 15% SDS-PAGE and detected with Coomassie Brilliant Blue R-250.
Western blotting analysis
Sup35NM proteins were analyzed by 15% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The membranes were blocked in 5% skim milk for 1 h in PBS buffer with 0.1% Tween 20 (PBST). Primary antibody (1:2500) was added and the membrane was incubated for 1 h, then washed with PBST. Secondary anti-rabbit antibody (1:5000) conjugated to horse-radish peroxidase was then added, and the membrane was incubated for 45 min. The proteins were visualized with enhanced chemiluminescence reagent. The development of the signal was recorded on photographic film.
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); rotating with 5 rpm (•); seeding with 2% preformed fibrils (
). At various times, duplicate aliquots were diluted to 2 µM protein and incubated together with 10 µM Congo red. (B) SDS-PAGE analysis of samples with Coomassie blue with 5 rpm rotating over time. (C) Western blots of the same samples probed by anti-M antibody. Aliquots were not heated (–) except for the last lane, which was heated (+) at 100°C for 10 min before the analysis. Mono, monomeric Sup35NM; Poly, Sup35NM amyloid fibrils.
