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Protein Science (2001), 10:854-863.
Copyright © 2001 The Protein Society

Strain-specified relative conformational stability of the scrapie prion protein

David Peretz1,2, Michael R. Scott1,2, Darlene Groth1,2, R. Anthony Williamson3, Dennis R. Burton3, Fred E. Cohen4,5,6 and Stanley B. Prusiner1,2,6

1 Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94143, USA
2 Department of Neurology, University of California, San Francisco, California 94143, USA
3 Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
4 Department of Medicine, University of California, San Francisco, California 94143, USA
5 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA
6 Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA

Reprint requests to: Stanley B. Prusiner, Institute for Neurodegenerative Diseases, Box 0518, University of California, San Francisco, CA 94143-0518; e-mail: hang{at}itsa.ucsf.edu; fax: (415) 476-8386.

(RECEIVED September 19, 2000; FINAL REVISION January 23, 2001; ACCEPTED January 23, 2001)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.39201


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrPSc). To explore this postulate, we examined the properties of PrPSc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrPSc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrPSc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have ~75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a ~170-d incubation period and ~1.25 M for the SHa(RML) and 139H isolates with ~180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a ~320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrPSc conformers encipher the biological properties of prion strains.

Keywords: Prion strains; spongiform encephalopathies; protein conformation; scrapie; prion protein; neurodegenerative disease


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Many lines of evidence show that the pathogenic protein (PrPSc) is the sole component of the infectious prion particle and that its formation derives from the post-translational modification of the cellular isoform (PrPC; Cohen and Prusiner 1998; Prusiner et al. 1998). Although the covalent structure of the two PrP isoforms appears identical (Stahl et al. 1993), they can be readily distinguished by their drastically different physical properties (Pan et al. 1993). PrPC is readily soluble in nondenaturating detergents, is rapidly digested by proteases, and is rich in {alpha}-helical structure and essentially devoid of ß-sheet content. In contrast, PrPSc is insoluble in such detergents, is resistant to proteolysis except for the N-terminal region comprising ~67 residues, and has a high ß-sheet content (Caughey et al. 1991; Gasset et al. 1993; Pan et al. 1993; Pergami et al. 1996; Safar et al. 1993). The protease-resistant fragment of PrPSc has a molecular size of 27–30 kD and is designated PrP 27–30 (Prusiner et al. 1983). It consists of ~142 amino acids and conveys prion infectivity. In the presence of detergent, PrP 27–30 readily polymerizes into amyloid although amyloid is neither obligatory for prion infectivity nor disease pathogenesis (Prusiner et al. 1983, 1990; McKinley et al. 1991). Protein denaturants abolish prion infectivity and protease resistance while increasing solubility and immunodetection of PrPSc (Kitamoto et al. 1987; Serban et al. 1990; Taraboulos et al. 1992; Prusiner et al. 1993; Oesch et al. 1994; Peretz et al. 1997; Safar et al. 1998). Thus, considerable evidence shows that prion diseases are disorders of protein conformation.

Prion strains have been shown to breed true on repeated passage in animals of the same species as by phenotypic characteristics including the clinical presentation of disease (Pattison and Millson 1961; Mastrianni et al. 1999), the length of the incubation period (Dickinson et al. 1968), the distribution of vacuolar degeneration (Fraser and Dickinson, 1968; Fraser 1979), and the pattern of PrPSc deposition in the CNS (Bruce et al. 1989; Hecker et al. 1992). The phenomenon of prion strains has been cited frequently as evidence that an independently replicating informational molecule or genome exists within the infectious particle (Bruce and Dickinson 1987). To accommodate multiple strains in the absence of a nucleic acid, PrPSc must be able to sustain separate information states within the same amino acid sequence, and PrPC must be able to faithfully acquire this information during its conversion into PrPSc. Substantial evidence suggests that a direct interaction between PrPC and PrPSc leads to the conversion of PrPC to PrPSc (Prusiner et al. 1990; Horiuchi et al. 1999). Accordingly, different strains must maintain different templates of PrPSc structures, and these differences at the molecular level ultimately should dictate strain properties.

Persuasive evidence that strain-specific information is enciphered in the structure of PrPSc arose from the transmission of two different inherited human prion diseases to mice expressing a chimeric human/mouse (MHu2M) PrP transgene (Telling et al. 1996). In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after enzymatic removal of the two N-linked glycans is 19 kD, whereas that from familial Creutzfeldt-Jacob disease (CJD) and sporadic CJD is 21 kD (Monari et al. 1994; Parchi et al. 1996). Extracts from the brains of patients with FFI transmitted disease to Tg mice and induced formation of the 19-kD PrPSc fragment; in contrast, extracts from the brains of patients with CJD produced the 21-kD PrPSc fragment in the same mice (Telling et al. 1996). These results showed that MHu2M PrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet, the amino acid sequence of MHu2M PrPSc remained invariant.

Although early comparisons of hamster prion strains did not reveal any particularly compelling biochemical differences in PrPSc (Kascsak et al. 1986; Hecker 1992), such differences were found for two transmissible mink encephalopathy (TME) prion strains, drowsy (DY) and hyper (HY), that were transmitted to hamsters (Bessen and Marsh 1992b). The DY strain was found to differ significantly from other known hamster prion strains in its biochemical and physical properties. Marked differences were identified by sedimentation analysis, protease sensitivity, and by the migration pattern of PrPSc proteolytic fragments on SDS gels (Bessen and Marsh 1992b). PrPSc comprising the DY prions showed diminished resistance to proteinase K digestion and yielded a protease-resistant fragment of 19 kD after deglycosylation, whereas that from HY was 21 kD (Bessen and Marsh 1994). Notably, TME strain properties could be preserved after transmission of either the full-length or the protease-resistant fragment of PrPSc, contending that strain characteristics are propagated by the protease-resistant core (Bessen and Marsh 1994).

Because most prion strains encipher PrPSc conformers that generally yield PrP 27–30 with a polypeptide core of 21 kD after limited proteolysis, it has not been possible to use the mobility-shift assay to detect plausible differences in PrPSc conformation in most cases (Bessen and Marsh 1994; Monari et al. 1994; Parchi et al. 1996; Telling et al. 1996; Scott et al. 1997). Moreover, the insolubility of PrPSc has prevented comparative structural studies of prion strains by using high-resolution nuclear magnetic resonance and X-ray diffraction techniques. Of note, Fourier-transform infrared spectroscopy of the HY and DY strains did give different spectra (Caughey et al. 1998). A conformation-dependent immunoassay (CDI) was used to investigate the eight SHa prion isolates studied here by quantification of the immunoreactivity of denatured (D) and native (N) PrPSc (Safar et al. 1998). The monoclonal antibody (mAb) 3F4 used in those studies recognizes a conformationally sensitive epitope (Peretz et al. 1997). In a plot of the D/N ratio as function of PrPSc concentration, each isolate occupied a unique position, a result consistent with the existence of multiple discrete PrPSc conformers that are strain-specified (Safar et al. 1998).

To test the hypothesis that the biological properties of prion strains are enciphered in the conformation of PrPSc, we examined the relative conformational stability of PrPSc derived from the SHa brains infected with eight strains. Because PrP 27–30, the protease-resistant core of PrPSc, is infectious and can initiate the faithful propagation of strains (Prusiner et al. 1983; Bessen and Marsh 1994), we studied the conformational stability of this molecule. Using sensitivity to protease as a marker for the denatured state of PrP 27–30, we characterized the protein conformations of eight hamster prion isolates. We found that these eight strains could separate into four groups based on relative conformational stability and incubation period. The sigmoidal shape of the conformational transition curves shows that the unfolding of PrPSc from a protease-resistant state to a sensitive one is a cooperative process. Our findings support the proposition that PrPSc can adopt multiple conformations. By inference, these results lend additional support to the hypothesis that the conformation of PrPSc enciphers the biological properties of prion strains.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Homogenates of brain from Syrian hamsters infected with Sc237, HY, and DY prion isolates were prepared when the animals displayed signs of CNS dysfunction. The protease-resistant core of PrPSc, denoted PrP 27–30, of the DY strain differs from that of Sc237 and HY with respect to migration on SDS-PAGE. PrP 27–30 from the brains of hamsters infected with the DY strain migrated slightly faster than the PrP 27–30 of either of the Sc237 and HY strains, and after 12 h of proteinase K digestion, much of the PrP 27–30 from the DY strain was hydrolyzed whereas the amounts of PrP 27–30 from the Sc237 and HY strains appeared unchanged (data not shown), consistent with previously published data (Bessen and Marsh 1992a; Scott et al. 1997). These observations suggested that the conformation of PrPSc specified by the DY strain is markedly different from that specified by the Sc237 and HY strains.

Guanidine hydrochloride denaturation of PrP 27–30
To extend the comparative analysis of the Sc237 and DY strains, we prepared a crude brain fraction by differential centrifugation and detergent extraction previously designated P2 (Prusiner et al. 1984). Aliquots of the P2 fraction were incubated with increasing concentrations of guanidine hydrochloride (GdnHCl) for 1 h followed by dilution of the GdnHCl and limited proteolysis (Fig. 1Go). By Western blotting using the 3F4 fragment antibody (Fab), the amount of PrP 27–30 for the Sc237 and DY strains remained unchanged up to 0.8 M GdnHCl. Exposure to 1.0 M GdnHCl did not alter the extent of digestion of PrP 27–30 of the Sc237 strain but caused a dramatic increase in the susceptibility to protease of PrP 27–20 of the DY strain. At higher concentrations of GdnHCl, the PrP 27–30 of the DY strain disappeared, and that of the Sc237 strain gradually diminished. Reductions in the amounts of PrP 27–30 from both strains were not followed by the appearance of protease degradation products, suggesting that once a PrPSc is rendered protease sensitive by GdnHCl, the entire protein is rapidly degraded. Moreover, we could find no evidence to suggest that degradation of PrP 27–30 of the Sc237 strain proceeded along a pathway in which PrP 27–30 of the DY strain was an intermediate. When the same study was repeated with the N-terminal D13 Fab and the C-terminal R1 Fab, similar patterns of PrP 27–30 stability for the Sc237 and DY strains were found (data not shown).



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  		Fig. 1. Western blot analysis of denaturation transitions for the Sc237 and DY strains. Ten microliters of denatured and proteinase K-treated protein samples used in ELISA has been analyzed by Western blotting using anti-PrP mAb 3F4, as described in Materials and Methods (Kascsak et al. 1987). Brain homogenates (10 mg/mL) were prepared from Syrian hamsters infected with Sc237 (S) and DY (D) prion isolates.

 
Relative conformational stabilities of the Sc237 and DY strains
To quantify the differences in the resistance of PrP 27–30 for the Sc237 and DY strains, we used ELISA. Proteins in the P2 fraction prepared from scrapie-infected hamster brains were denatured with increasing concentrations of GdnHCl before limited digestion with proteinase K. As shown in Figure 2AGo, the amount of PrP was constant over the broad range of GdnHCl concentrations (0–3 M) when proteolytic digestion was omitted for hamsters infected with Sc237 or DY.



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  		Fig. 2. ELISA of denaturation transitions for the Sc237 and DY strains. (A) P2 fractions prepared from brains of normal Syrian hamster (black bars), Sc237 (white bars), and DY (striped bars) were treated with GdnHCl and precipitated with methanol/chloroform, and ELISA wells were coated with 50 µL of ~10 µg/mL of proteins. Proteins were denatured in situ with 3 M GdnSCN, and PrP was detected with mAb D18 (1 µg/mL). (B, C) After 1 h of denaturation, the samples were diluted and treated with PK for 1 h at 37°C. ELISA wells were coated with 50 µL of 3 (diamonds), 6 (circles), 12 (triangles), and 25 (squares) µg/mL of denatured proteins. PrP was detected with D18 Fab, which recognizes the epitope within residues 133–157 (Williamson et al. 1998). The sigmoidal patterns of Sc237 PrP (filled symbols in B) and DY PrP (open symbols in C) were plotted with a four-parameter algorithm by using a nonlinear least-squares fit and correlation coefficient >0.97. (D) The apparent fractional appearance (fapp) of Sc237 (filled symbols) and DY PrP (open symbols) were calculated for each O.D. and fitted for each antigen concentration, as described in Materials and Methods. Each data point shown is the mean of duplicate readings and error bars represent SEM.

 
Consistent with Western blot analysis (Fig. 1Go), the amount of PrP 27–30 of the Sc237 strain was inversely proportional to the concentration of GdnHCl (Fig. 2BGo). Similar findings were obtained when PrP 27–30 of the DY strain was examined (Fig. 2CGo). Moreover, the results for both strains were relatively independent of the protein concentration as shown in a plot in which the amounts of PrP 27–30 for the two strains were normalized (Fig. 2DGo). The amounts of PrP 27–30 from both strains as a function of the GdnHCl concentration showed sigmoidal transitions. For the Sc237 strain, the level of PrP 27–30 remained unchanged up to ~1.0 M GdnHCl, whereas for the DY strain, it was constant up to ~0.6 M. Once the levels of PrP 27–30 began to decrease, the diminution was progressive and approached zero at a concentration of ~2.0 M GdnHCl for Sc237 and ~1.5 M for DY.

The most reliable interpolation of values for such sigmoidal curves occurs in the middle portion between the two asymptotes, that is, the inflection point, of the curve rather than farther along the asymptotes (Rodgers 1984). Therefore, we used the GdnHCl concentration found at the half-maximal denaturation (GdnHCl1/2) as a measure of the relative conformational stability of PrPSc. Consistently, we found that coating the ELISA plates with 25 µg/mL of protein results in lower signals compared with those obtained with 12.5 µg/mL protein (Fig. 2B,CGo). That coating with high protein concentrations leads to reductions in final signals is well documented, and at least two different mechanisms have been suggested to explain this phenomenon (Cantarero et al. 1980). Therefore, measurements of GdnHCl1/2 values were obtained from ELISA plates coated with 10 µg/mL of protein. At these protein concentrations, the mean GdnHCl1/2 values for PrP 27–30 of the DY and Sc237 strains were 1.08 M and 1.47 M, respectively (Table 1Go).


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  		Table 1. Nomenclature and characteristics of SHa prion strains
 
Relative conformational stabilities of additional hamster prion strains
Besides Sc237, six other prion strains propagated in Syrian hamsters were found to be indistinguishable from Sc237 with respect to the migration of PrP 27–30 on SDS-PAGE and sensitivity to protease digestion (Hecker et al. 1992; Scott et al. 1997). When these six additional strains were denatured with increasing concentrations of GdnHCl, all showed sigmoidal patterns of conformational stability (Fig. 3Go). Like Sc237, the amounts of PrP 27–30 were unchanged after treatment with concentrations of GdnHCl up to ~1.0 M for the HY, SHa(ME7), and MT-C5 strains. The GdnHCl1/2 values for these four strains ranged from 1.47 M to 1.5 M and were not statistically different from each other. In contrast, treatment of the 139H and SHa(RML) strains with 1.0 M GdnHCl resulted in denaturation of ~25% of the PrP 27–30. The GdnHCl1/2 values for the SHa(RML) and 139H strains were 1.25 M and 1.26 M GdnHCl, respectively. The GdnHCl1/2 value for the ME7-H strain was 1.39 M GdnHCl.



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  		Fig. 3. ELISA of denaturation transitions for six prion strains. P2 fractions prepared from a pool of five brains were treated with GdnHCl, PK digested, and precipitated with methanol/chloroform as previously described. ELISA wells were coated with 50 µL of ~10 µg/mL of proteins, and PrP was detected with anti-PrP D18 Fab. Sigmoidal patterns of PrPSc strains were plotted with correlation coefficient >0.97, and Fapp values were calculated for each O.D. Each symbol represents a separate experiment.

 
To test differences between individual isolates and to separate the isolates into groups with P values <0.05, we used Tukey's method for multiple comparisons procedure with SAS (Statistical Analysis System, version 8.0; SAS Institute). Seven of the eight isolates could be separated into three distinct groups: group 1: SHa(Me7), MTC-5, Sc237, HY; group 2: 139H, SHa(RML); group 3: DY. The remaining isolate, Me7-H, was distinct from groups 2 and 3 and overlapped slightly with Sc237 and HY but not SHa(Me7) and MTC-5 from group 1 (Table 1Go). When we repeated this procedure with P values <0.1, complete separation into four groups was observed.

The similar GdnHCl1/2 values observed for PrP 27–30 molecules of the four strains Sc237, HY, SHa(ME7), and MT-C5 are notable; moreover, these four strains display incubation periods of ~75 days (Fig. 4Go; Table 1Go). Interestingly, the GdnHCl1/2 values found for the 139H and SHa(RML) strains were similar, and both these strains have incubation times of ~180 days. In contrast, the DY strain, with an incubation time similar to that of the 139H and SHa(RML) strains, has a strikingly different GdnHCl1/2 value. It is noteworthy that size of the deglycosylated PrP 27–30 polypeptide is 19 kD for the DY strain and 21 kD for the 139H and SHa(RML) strains as well as the other five strains analyzed in this study. The Me7-H strain had an incubation time of ~320 days and a GdnHCl1/2 value of 1.39 M, indicating that there is no quantitative relation between the length of the incubation period and the conformational stability of PrP 27–30 (Fig. 4Go). Notably, Me7-H, Sc237, and HY had GdnHCl1/2 values that were not significantly different at P < 0.05 (Table 1Go), suggesting that relative conformational stability alone cannot always used to discriminate strains.



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  		Fig. 4. Incubation period and GdnHCl1/2 values for eight SHa strains. ELISA wells were coated with ~0.5 µg of P2 proteins and GdnHCl1/2 values were interpolated from at least four denaturation curves. Each data point shown is the mean, and error bars represent standard deviation.

 
Recombinant anti-PrP Fabs
Although the foregoing ELISA results were obtained with the recombinant D18 Fab that binds to residues 133–152, recent work has produced a series of recombinant Fabs to a variety of epitopes across the entire PrP molecule (Williamson et al. 1996, 1998; Peretz et al. 1997). To investigate further the conformational stability of PrPSc, we studied the denaturation transitions of PrP 27–30 by using the D13, 3F4, and R2 Fabs that bind epitopes delimited by residues 96–106, 109–112, and 225–231, respectively (Peretz et al. 1997). Despite the differences in the affinities and binding sites of these Fabs, all the Fabs gave GdnHCl1/2 values similar to that found with the D18 Fab for the Sc237 strain (Table 2Go). The three Fabs gave slightly higher GdnHCl1/2 values than that found with the 3F4 Fab for the DY strain (Table 2Go). These data are consistent with the proposal that the transition from native to denatured PrPSc follows a cooperative pathway that is strain specific.


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  		Table 2. [GdnHCl]1/2 values for different regions of Sc237 and DY PrP
 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
For more than a decade, the prion concept encountered considerable skepticism largely because strains of prions demanded an informational macromolecule. Before the discovery of prions, such macromolecules were always nucleic acids. That different forms of PrPSc might explain strains of prions seemed remote especially because no covalent change was found that differentiated PrPSc from PrPC. The drastically different conformations that were found to distinguish PrPSc from PrPC raised the possibility that the biological properties of prion strains are specified by the tertiary structure of PrPSc. Transmission of prions from patients with familial CJD (E200K) and FFI to mice expressing MHu2M PrP transgenes showed that prions from these well-defined sources produced MHu2M PrPSc molecules with distinct conformations in the same line of mice (Telling et al. 1996). These studies were particularly compelling because each inoculum was derived from a patient with an inherited prion disease that was caused by a specific mutation in the PrP gene. Our findings and earlier studies with two strains of prions isolated from mink showed that PrPSc can exist in at least two conformations based on migration of PrP 27–30 on SDS-PAGE (Bessen and Marsh 1994).

The foregoing findings set the stage to ask how many conformations PrPSc can adopt. Before the finding that PrPC and PrPSc have the same covalent structure but possess different conformations, it generally was accepted that a particular amino acid sequence adopts only one biologically active conformation (Anfinsen 1973). To explore the range of biologically active conformations that PrPSc can adopt, we chose two different approaches. First, we developed the CDI, a new assay for PrPSc that does not require proteolytic digestion of PrPC before measuring PrPSc. The CDI not only measures the precursor of PrP 27–30, which is denoted rPrPSc, but it also measures an intermediate in prion replication, denoted sPrPSc for a protease-sensitive form of PrPSc (Safar et al. 1998). The levels of both rPrPSc and sPrPSc were found to be specified by the prion strain, but only the level of sPrPSc showed a correlation with the strain-specified disease characteristic, that is, the level of sPrPSc was directly proportional to the length of the incubation time. Second, we developed a conformational stability assay for PrP 27–30 that is more discriminating than limited proteolysis and migration on SDS-PAGE, as reported here.

Measurements of the conformational stability of soluble proteins have shown that slight differences in protein structure can be measured by exposure to a denaturant over an appropriate range of concentrations (Shirley 1995). These studies require the determining of the equilibrium constant and the free energy change, {Delta}G, for the reaction folded - unfolded. Because these are thermodynamic values, it is necessary that the unfolding reaction has reached equilibrium and that the unfolding reaction is reversible. Based on such findings, we developed a procedure in which PrPSc in crude fractions is exposed to GdnHCl and then digested with proteinase K. As the concentration of GdnHCl increased, the digestion of PrP 27–30 increased concomitantly. However, in experiments involving insoluble oligomeric forms of PrPSc, denaturation reversibility is not attainable, and therefore we did not calculate the {Delta}G of PrPSc. Alternatively, we chose to use the GdnHCl concentration found at the half-maximal denaturation (GdnHCl1/2) as a measure for the relative conformational stability of PrPSc. As reported here, the procedure yielded highly reproducible curves that, with incubation time to disease, could discriminate four different groups of strains from the eight that were studied.

We found that each strain had a characteristic relative conformational stability and that, in some cases, differences in relative stability were of sufficient magnitude that strains could be clearly discriminated based on this criterion alone. For example, the GdnHCl1/2 value of the DY strain (~1.0 M) was markedly different from that of the HY strain (~1.5 M) and Me7-H (1.4 M) isolates. Similarly, the SHa(RML) and 139H strains, with GdnHCl1/2 values of ~1.25 M, could be readily discriminated from DY (~1.0 M) although all three have similar incubation periods. It was also possible to distinguish the SHa(RML) and 139H strains from the SHa(Me7), Me7-H, HY, Sc237, and MT-C5 strains even though these strains could not be discriminated using the SDS–gel mobility-shift assay (Scott et al. 1997). Thus, relative conformational stability may provide a useful biochemical marker for strain typing. To put this proposal in context, the recent demonstration that BSE could be experimentally transmitted to sheep has raised considerable concern in the United Kingdom that sheep exposed to contaminated animal feed might be harboring BSE (Foster et al. 1993). Perhaps a comparison of the relative conformational stability of sheep-passaged BSE and natural sheep scrapie prions might allow a simple diagnostic test suitable for screening the British sheep population for the presence of BSE to be designed.

Notably, we also found that some strains, previously known to be distinct by other criteria, such as Sc237 and Me7-H, had relative conformational stabilities that were similar (~1.5 M vs. ~1.4 M GdnHCl). These results suggest that the criterion of relative conformational stability alone cannot always be used to discriminate strains. However, even in such instances, relative stability may be used to complement other phenotypic criteria when comparing prion strains. When the eight prion isolates were analyzed by the dual criteria of incubation period and relative conformational stability (Fig. 4Go), it became clear that the eight strains could be segregated into only four clusters of strains.

No quantitative relation between the length of the incubation time and the degree of conformational stability could be discerned (Fig. 4Go). Before this study, it was certainly a possibility that variations in incubation period seen with prion strains might be caused by variation in the stability of PrP 27–30, but our new work clearly shows that this is not the case. We interpret these findings to suggest that strain characteristics, like rate of PrPSc formation as well as sites of replication and accumulation, ultimately might be more important in determining incubation time to disease. It is noteworthy that no relation between the level of PrP 27–30 and the length of the incubation time could be found for these same eight strains using the CDI; however, the level of sPrPSc was directly proportional to the length of the incubation time (Safar et al. 1998). The conformational stability assays reported here cannot be compared directly with this finding because they have not been performed on sPrPSc.

Based on our conformational stability assays, it can be argued that Sc237, HY, SHa(ME7), and MT-C5 are a single strain of prion. They have the same incubation times in hamsters, and they show indistinguishable conformational stability curves (Table 1Go; Fig. 3Go). When the distribution of vacuolation and the pattern of PrPSc deposition in brains of Syrian hamsters inoculated with SHa(RML), SHa(Me7), Me7-H, and Sc237 strains were compared, only the Me7-H strain was markedly different by both criteria (Scott et al. 1997). Also, whereas SHa(RML), SHa(Me7), and Sc237, distinct from Me7-H, the SHa(RML) strain gave a pattern of deposition that could be discriminated easily by histoblotting from the SHa(Me7) and Sc237 strains, which showed only minor differences in the intensity and distribution of the PrPSc signal (Scott et al. 1997). Therefore, based on multiple criteria of incubation period, neuropathology, and PrPSc conformational stability, it certainly is possible that at least the SHa(Me7) and Sc237 strains represent a single strain of prion. However, note that both of these strains, together with HY and MT-C5, could be discriminated by CDI in plots of the concentration of PrPSc as a function of the D/N ratio (Safar et al. 1998). Whether these are truly four strains as can be argued from the CDI data or they are one strain based on incubation times and conformational stabilities remains to be established. The same questions attend to the SHa(RML) and 139H strains, with ~180-d incubation periods and similar conformational stabilities.

The sigmoidal shape of the GdnHCl denaturation curves was similar for all eight strains examined (Figs. 2 and 3GoGo); furthermore, the shape of the curves was unaltered when a variety of recombinant Fabs directed against three different epitopes were used in addition to the 3F4 Fab (Table 2Go). Additionally, the sigmoidal shape of the denaturation curves was independent of the concentration of protein used in the assay. These findings show that unfolding of PrP 27–30 is a cooperative process.

Previously, a comparison of the reactivity of antibodies to PrPC with those to PrPSc dispersed in detergent–lipid–protein complexes revealed that the major conformational differences between the two molecules lie within the N-terminal region of PrP 27–30 corresponding to residues 90–120 (Peretz et al. 1997). Whether this local conformational change alone can explain the protease resistance of residues 90–231 comprising PrP 27–30 or whether oligomerization plays an important role remains to be established. Results of ionizing radiation inactivation experiments show that the minimal infectious unit may be a dimer of PrPSc (Bellinger-Kawahara et al. 1988), suggesting that quaternary packing of PrPSc monomers might contribute to the structural change that confers the resistance of PrP 27–30 residues to protease digestion.

Although we have chosen to interpret the conformational stability curves in terms of a two-state process, that is, the transition from the native to the denatured state, we recognize that the events that make up this transition are far more complicated. Under the conditions of our experiments at 0 M GdnHCl, it is likely that the digestion of rPrPSc produces PrP 27–30 that rapidly polymerizes into rod-shaped structures with the properties of amyloid. As the concentration of GdnHCl is raised, it is unclear when the polymerization process ceases to occur. Whether polymerization of PrP 27–30 into amyloid is more sensitive to disruption by GdnHCl than the events that render PrP 27–30 susceptible to proteolysis is unknown.

The results presented here clearly show that PrPSc may adopt at least three different conformations. Each of these conformations is associated with a distinct disease phenotype suggesting that biological properties of each strain are enciphered in the tertiary and possibly quaternary structure of PrPSc. The mechanism by which prions replicate with a high degree of fidelity and thus are able to preserve the strain-specified properties is unknown. The existence of multiple prion strains (Table 1Go; Fig. 4Go) demands that the conformation of PrPSc be faithfully copied during prion replication. Some of us have suggested that such a process involves a template-assisted process in which one or more auxiliary molecules provisionally designated protein X facilitate the production of nascent PrPSc (Telling et al. 1995; Kaneko et al. 1997; Zulianello et al. 2000). An alternate proposal suggests that prion replication occurs through a nucleation–polymerization process (Gajdusek 1988; Jarrett and Lansbury 1993; Caughey and Chesebro 1997), but against this proposal is the finding that full-length PrPSc does not form regular polymers (McKinley et al. 1991) and prion replication is restricted to a few tissues although PrPC is much more widely expressed (Raeber et al. 1999). Only PrP 27–30 and smaller fragments of PrP form regular polymers with the ultrastructural and tinctorial features of amyloid (Prusiner et al. 1983; Gasset et al. 1992; Forloni et al. 1993); moreover, amyloid polymers composed of PrP 27–30 are clearly not obligatory for infectivity (Gabizon et al. 1987; McKinley et al. 1991; Wille et al. 1996). Elucidating the mechanism of PrPSc formation is likely to be important in defining the limits of prion diversity.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Scrapie prion strains
The Sc237, HY, and DY hamster PrP strains were a gift from Dr. Richard Marsh (Marsh and Kimberlin 1975) and were repeatedly passaged in golden Syrian hamsters (LVG:Lak) purchased from Charles River Laboratory. The MT-C5 strain was isolated in SHa (LVG:Lak) from a cow infected with a second passage of sheep scrapie (Gibbs et al. 1996). The Me7-H and 139H isolates were generously donated to us by Drs. R. Kimberlin and R. Carp (Department of Virology, New York Institute for Basic Research in Developmental Disabilities). The SHa(ME7) and SHa(RML) strains were generated in our laboratory by passage of the Me7 (Zlotnik and Rennie 1965) and RML (Chandler 1961) isolates from mice to transgenic mice expressing a chimeric mouse/hamster (MH2M) PrP, followed by two successive passages in Syrian hamsters (Scott et al. 1997). Although the SHa(Me7) and Me7-H isolates are distinct when assessed by incubation period and neuropathology (Scott et al. 1997), they were both derived from the same ancestral mouse scrapie strain, Me7, which originally was derived by passage of natural Suffolk sheep scrapie to mice (Zlotnik and Rennie 1965). Subsequent transmission of Me7 from mice directly to Syrian hamsters led to the isolation of the Me7-H strain (Kimberlin et al. 1989). Animals were inoculated intracerebrally with 50 µL of 1% brain homogenate containing scrapie prions as described (Scott et al. 1997). The diagnosis of scrapie and the killing of hamsters have been described previously (Prusiner et al. 1982, 1990; Carlson et al. 1986).

Preparation of protein fraction P2
P2 fraction was prepared as previously described with slight modifications (Prusiner et al. 1984). Briefly, brains were homogenized (10% w/v) in 320 mM sucrose. The homogenate was centrifuged at 4000g for 30 min at 4°C. To the supernatant, we added Triton X-100 and DOC at detergent/protein (w/w) ratios of 4:1 and 2:1, respectively, and mixed for 1 h at 4°C. To the detergent extract, we added glycerol and polyethylene glycol (PEG) and mixed for 1 h at 4°C as described (Prusiner et al. 1984). The PEG precipitate was collected by centrifugation at 10,000g for 30 min at 4°C. The pellet, termed P2 (Prusiner et al. 1984), was resuspended in 20 mM Tris-acetate at pH 8.3 containing 0.02% Triton X-100 and 1 mM dithiothreitol and adjusted to a protein concentration of 10 mg/mL.

Expression and preparation of anti-PrP mouse Fabs
The preparation of Fabs libraries from PrP gene knockout mice (Prnp0/0) immunized with liposomes containing dispersed SHaPrP 27–30 has been described (Peretz et al. 1997). Selected mouse Fab clones (i.e., D13, D18, and R2) were grown in quantity, and the Fabs were affinity-purified using polyclonal goat anti-mouse IgG Fab (Pierce) covalently bound to protein G matrix (Pharmacia) as described (Williamson et al. 1993). The 3F4 Fab fragments were prepared from hybridoma-derived 3F4 mAb (Kascsak et al. 1987) treated with the endopeptidase enzyme papain (Pierce).

Western blot analysis
An equal volume of 2 x sample buffer (Laemmli 1970) was added to protein samples. Each SDS-PAGE lane was loaded with 10 µg of total protein. PrP was detected with anti-PrP 3F4 monoclonal antibodies (1 µg/mL; Kascsak et al. 1987), and enhanced chemiluminescence detection method (Amersham Corporation).

ELISA denaturation transition
Aliquots of 25 µL from the P2 fraction (10 mg/mL) were mixed with 25 µL of GdnHCl stock solutions, with final GdnHCl concentrations ranging from 0 M to 4 M. GdnHCl stock solutions were prepared from an 8-M solution (Pierce) diluted in water. After 1 h of incubation at room temperature, all samples were diluted with phosphate-buffered saline to a final concentration of 0.1 M GdnHCl and 2% Sarkosyl in a volume of 1.5 mL. Proteinase K (PK) (0.5 µg) was added, and the samples were incubated for 1 h at 37°C. The reaction was stopped with 2 mM PMSF and a cocktail of protease inhibitors (Boehringer Mannheim). Proteins were precipitated with 4 volumes of methanol/chloroform (2:1 v/v) for 14 h at -20°C. Samples were centrifuged at 4000g for 30 min at 4°C. Pellets were resuspended in 50 µL of 6 M GdnSCN solution for 1 h and diluted into 1.5 mL of ELISA binding buffer (0.1 M bicarbonate at pH 8.6). ELISA wells were coated with 50 µL of solution containing 10 µg/mL of protein. To increase the immunoreactivity of PrPSc, we denatured coated proteins in situ with 50 µL of 6 M GdnSCN. After 10 min at room temperature, the plates were washed three times and blocked, and immunocomplexes of Fab-PrP were detected as described (Burton et al. 1991). PK was fully active in the presence of 0.1 M GdnHCl as measured by a colorimetric assay with carbobenzoxy-valyl-glycyl-arginine p-nitroanilide (Boehringer Mannheim).

Data analysis of ELISA denaturation transition curves
After addition of the color development substrate p-nitrophenyphosphate, ELISA plates were incubated for 1 h at 37°C, and color absorbance was measured at 405 nm by using spectrophotometer V-max (Molecular Devices). P2 samples prepared from normal SHa brains, proteinase K digested and methanol/chloroform precipitated, were used as blanks (O.D. value of ~0.1). The O.D. patterns were best fitted using the four-parameter sigmoid equation (Maquard-Levendberg algorithm) using the software SigmaPlot (SPSS).

To plot the remaining fraction of PrPSc as a function of GdnHCl concentration, we expressed each measured O.D. (O.D.mean) within the curve as the apparent fractional extent of native PrPSc, given that at maximum fitted O.D. (O.D.max), all PrPSc is present, with a value of 1, and at O.D. close to zero (O.D.min), most of PrPSc is denatured and degraded by PK. The following equation was used (Kuwajima 1995): .


   Acknowledgments
 
This work was supported by a research grant from the National Institutes of Health (NIH AG10770) as well as gifts from the G. Harold and Leila Y. Mathers Foundation, the Sherman Fairchild Foundation, the Bernard Osher Foundation, and Centeon. David Peretz was supported by a postdoctoral fellowship from the John Douglas French Foundation for Alzheimer's Disease.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


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 Materials and methods
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