Analyses of Protease Resistance and Aggregation State of Abnormal Prion Protein across the Spectrum of Human Prions

Background: Protease resistance and aggregation state, two fundamental properties of abnormal prion protein (PrP^Sc), remain significantly unexplored.

Results: Prion isolates show differences in PrP^Sc PK digestion profile that are only partially explained by differences in aggregate size.

Conclusion: Molecular factors, likely acting on aggregate stability, significantly influence PrP^Sc PK sensitivity.

Significance: The study provides insight into prion strain variability with respect to PrP^Sc PK sensitivity and aggregation state.

Abstract

Prion diseases are characterized by tissue accumulation of a misfolded, β-sheet-enriched isoform (scrapie prion protein (PrP^Sc)) of the cellular prion protein (PrP^C). At variance with PrP^C, PrP^Sc shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrP^Sc itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrP^Sc (sPrP^Sc) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrP^Sc, however, remains technically challenging because of the need of separating it from PrP^C without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrP^Sc across the whole spectrum of the currently characterized human prion strains. The results show that PrP^Sc isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrP^Sc aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrP^Sc (i.e. <10%), arguing against a significant role of slowly sedimenting PK-sensitive PrP^Sc in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrP^Sc and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrP^Sc form.

Introduction

Prion diseases or transmissible spongiform encephalopathies (TSEs)² are a phenotypically heterogeneous group of rare neurodegenerative disorders affecting humans and other mammals. These diseases are characterized by tissue deposition of a misfolded form of the endogenous cellular prion protein (PrP^C), a membrane glycosylphosphatidylinositol-anchored glycoprotein of 231 amino acids comprising three glycoforms (i.e. diglycosylated, monoglycosylated, and unglycosylated) that are generated by the non-obligatory addition of N-linked glycan chains at residues Asn-181 and Asn-197 (1). Human PrP^C is composed of an unstructured N-terminal domain followed by a well characterized globular domain that comprises two β-sheets and three α-helices (2). Although it is well established that PrP^C conversion into its misfolded form, PrP^Sc, involves consistent changes in the secondary structure with part of the α-helical structure turning into a β-sheet (3, 4), a further structural characterization of PrP^Sc has been hampered to date by the propensity of the misfolded protein to form highly aggregated and detergent-insoluble polymers.

Human prion diseases are characterized by a number of clinicopathological types or variants. Different strains of the TSE agent or prion are believed to be the main cause of this phenotypic diversity (5). In addition, the host variability in the gene encoding PrP (PRNP) as determined in humans by polymorphisms or mutations also modulates the disease phenotype (6–8). In sporadic Creutzfeldt-Jakob disease (sCJD), the most common human TSE, six major disease phenotypes have been characterized that strongly correlate at the molecular level with the genotype at the polymorphic codon 129 (methionine (M) or valine (V)) of PRNP and two PrP^Sc profiles or types (type 1 and type 2) comprising distinctive physicochemical properties such as size after protease treatment and glycoform ratio (9, 10). The six sCJD types differ among each other in several clinical and histopathological features, and at least three of them (MM1, VV2, and MM2-thalamic (MM 2T)) propagate in animal models as distinct prion strains (11–14). Besides sCJD, a novel form of human sporadic prion disease designated variably protease-sensitive prionopathy (VPSPr) has been recently identified. When compared with sCJD, VPSPr displays “atypical” histopathologic and clinical features and shows a quite distinctive Western blot profile of abnormal PrP comprising truncated fragments in the 8–17 molecular weight range that are not seen in classic CJD (15). Finally, variant CJD (vCJD), an acquired form of the disease that originated from a bovine prion strain, also displays a very distinctive CJD phenotype (16, 17). PrP^Sc extracted from vCJD brains is biochemically distinguishable from the type 2 found in sCJD due to a relatively high representation of the diglycosylated PrP^Sc glycoform (18, 19). To date, only individuals that are methionine homozygotes in PRNP codon 129 have been affected by this CJD phenotype (20).

A central issue in prion biology concerns the molecular basis of prion strains. It is currently widely believed that prion strain diversity is encoded in the PrP^Sc conformation (1). Support for this idea comes from several studies showing that PrP^Sc exists in a number of molecular types that differ in one or more of the following properties: fragment size after protease digestion, glycoform profile, aggregate size, and stability in chaotropes (for a review, see Ref. 21). However, as this evidence is largely indirect, the structural basis of this heterogeneity remains to be established.

Although resistance to proteinase K (PK) and detergent insolubility have been traditionally used to operationally define PrP^Sc and to distinguish it from PrP^C, the recent characterization of a PK-sensitive form of PrP^Sc (sPrP^Sc) has highlighted a more complex picture and added a new perspective to the study of PrP^Sc properties and their relationship to prion strains (22–25).

Following the observation that in a minority of subjects affected by Gerstmann-Sträussler-Scheinker disease linked to the P102L mutation detergent-insoluble full-length PrP^Sc is easily digested at a relatively low PK concentration (26), similar PK-sensitive forms of PrP^Sc have been found in other rare, atypical variants of both human and animal prion diseases (15, 27–30).

However, by showing that the denaturation-dependent immunoreactivity enhancement effect that is attributed to PrP^Sc is considerably weakened by a pretreatment with PK, Safar et al. (24, 31) have proposed that sPrP^Sc does not only characterize atypical prions but rather constitutes an invariable and quantitatively significant component of prions, contributing up to 90% of the whole PrP^Sc signal even in TSEs such as sCJD and classical scrapie. With this novel approach, called conformation-dependent immunoassay (CDI), a correlation between the relative amount of sPrP^Sc and strain-specific properties such as the incubation period after inoculation and the clinical duration of the disease has also been reported (24, 25, 32). These findings have on one hand called into question the rationale on which TSE diagnosis is currently based (i.e. demonstration of rPrP^Sc) (24, 33, 34) and on the other supported the idea that sPrP^Sc contributes to the molecular basis of phenotypic variability and strain diversity in both animal and human prion diseases.

Concerning the physical nature of sPrP^Sc with respect to its “resistant” counterpart, the isolation and characterization of a sPrP^Sc fraction consisting of aggregates of shorter size with respect to rPrP^Sc (22, 23) has suggested that the resistance of PrP^Sc to proteolysis may, at least in part, depend on its quaternary structure. However, these PK-sensitive aggregates with low sedimenting properties are unlikely to reflect the whole sPrP^Sc signal defined by CDI because low sedimenting PrP^C-rich fractions contain little or no conformation-dependent immunoreactive material (22, 23), thus leaving the structural basis of the CDI-related sPrP^Sc signal unexplored. Furthermore, PrP^Sc associated with atypical animal and human prion diseases such as Nor98 or subtypes of Gerstmann-Sträussler-Scheinker disease did not exhibit a higher proportion of slowly sedimenting species when compared with classic strains (29, 35), arguing that, at least in these atypical prionopathies, the pronounced PK sensitivity of PrP^Sc is not due to low size aggregates but rather to its tertiary structure or other factors.

To further address these issues and gain insights into the molecular basis of phenotypic variability and disease pathogenesis in human prion disease, in the present study we have characterized the detergent-insoluble PrP^Sc fraction in relation to both PK resistance and the aggregation state across the whole spectrum of the currently characterized human prion strains. In particular, for each strain or disease subtype we have searched for and quantified the low sedimenting sPrP^Sc fraction, defined the sedimenting velocity profile of PrP^Sc, studied the overall resistance of PrP^Sc to PK digestion, and finally compared the resistance of PrP^Sc to proteolysis between strains in fractions with similarly sized aggregates.

EXPERIMENTAL PROCEDURES

Patients and Tissues

We have studied brain tissues from 41 cases of sCJD, three cases of vCJD, and two cases of VPSPr, the latter carrying, respectively, MM and MV at PRNP codon 129. sCJD tissues comprised the whole spectrum of pure phenotypic variants described by Parchi et al. (9) and included eight MM1, eight VV1, seven MV 2K, eight VV2, six MM2-cortical (MM 2C), and four MM 2T cases (9). In addition, three brains lacking significant histopathological changes were used as prion negative controls. Brain tissues were obtained at autopsy and kept frozen at −80 °C until use. All samples used in this study were taken from the frontal cerebral cortex.

Preparation of Total Brain Homogenates (THs)

50 mg of gray matter were homogenized (10%, w/v) in LB100, a lysis buffer with high buffer capacity (100 mm Tris, 100 mm NaCl, 10 mm EDTA, 0,5% Nonidet P-40, 0,5% sodium deoxycholate) at pH 6.9 (36). Total protein concentration was measured using a standard colorimetric method based on bicinchoninic acid (Pierce).

PrP^Sc Purification

Purified PrP^Sc was obtained from about 500 mg of gray matter after three cycles of Sarkosyl extraction and differential centrifugation steps (adapted from Ref. 37). Briefly, tissues were homogenized in 1.75 ml of 2× TEND (20 mm Tris, pH 8.3, 1 mm EDTA, 130 mm NaCl, 1 mm dithiothreitol) and 1.75 ml of 20% Sarkosyl and cleared by ultracentrifugation at 22,500 × g (Beckman rotor SW55Ti) for 25 min at 4 °C. Supernatants (S1) were then underlaid with a sucrose cushion (1 ml of 20% sucrose in H₂0) and ultracentrifuged at 150,000 × g for 143 min at 4 °C. After discarding the supernatants (S2), the pellets (P2) were incubated overnight at 4 °C in 100 μl of TBS and then resuspended in 2.5 ml 1× TEND, 10% NaCl, and 1% Sarkosyl through three cycles of freezing and thawing after a short and standardized sonication. Samples were then again laid over a sucrose cushion and ultracentrifuged at 150,000 × g for 143 min at 18 °C. After removal of the supernatant (S3), pellets (P3) were resuspended in 400 μl of TNS (10 mm Tris, 150 mm NaCl, 1% Sarkosyl), pH 7.4; incubated overnight; and then dissolved through three cycles of freezing and thawing after a short and standardized sonication.

Sucrose Gradient Centrifugation Assay

Total protein concentration in P3s was adjusted to a final value of 300 μg/ml. S2s and P3s (400 μl) were loaded on top of a 10–60% sucrose step gradient (e.g. 500 μl of 10, 20, 30% 40, 50, and 60% solutions of sucrose in TNS, pH 7.4 going from the top to the bottom of the tube) and ultracentrifuged at 200,000 × g for 1 h at 4 °C. After centrifugation, 12 fractions of 280 μl were collected from the top to the bottom of the tube.

PK Titration Assays

P3s were adjusted to a total protein concentration of 150 μg/ml and digested using serial dilutions of PK activity ranging from 0.004 to 20 units/ml. Because of the higher protein content, the total protein concentration of THs was adjusted to 6 mg/ml, whereas the PK activity used ranged from 0.03 to 256 units/ml.

Finally, the selected fractions (6, 9, and 12) from MM1 and VV2 cases were adjusted to a total protein concentration of 50 μg/ml and a sucrose content of 20%, whereas the range of PK dilutions varied from 0.016 to 40 units/ml. PK digestion was performed for 1 h at 37 °C unless otherwise specified.

PrP Deglycosylation

N-Linked glycans were removed by using a peptide-N-glycosidase F kit (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions.

Western Blot and Quantitative Analysis of Protein Signal

After the addition of sample buffer (final concentration, 3% SDS, 4% β-mercaptoethanol, 10% glycerol, 2 mm EDTA, 62.5 mm Tris, pH 6.8) and boiling for 6 min at 100 °C, samples were run on 13 or 15% (only for PK digestion experiments on THs) acrylamide gels (Criterion, Bio-Rad) and transferred to Immobilon-P membranes (Millipore Corp., Billerica, MA). After blocking in 10% nonfat milk in Tween-Tris-buffered saline, membranes were incubated with mAb SAF 60 (working dilution, 1:2000), which maps on residues 157–161 of the prion protein or with mAb 3F4 (Signet Laboratories, Dedham, MA; working dilution, 1:30,000), which maps on residues 106–110.

After four washings in Tween-Tris-buffered saline, membranes were incubated for 1 h with an anti-mouse secondary antibody conjugated to horseradish peroxidase (GE Healthcare; working dilution, 1:4000) and washed again four times in Tween-Tris-buffered saline. The immunoreactive signal was detected by enhanced chemiluminescence (ECL; GE Healthcare) on an LAS 3000 camera (Fujifilm Corp., Tokyo, Japan) or (only for the PK digestion experiments on THs) on Kodak Biomax Light films (Eastman Kodak Co.).

To quantify PrP content, the Western blot signal of each sample was compared with a standard curve obtained by loading in each gel the same serial dilution (n = 6) of a chosen sCJD sample. Western blot signals were measured by densitometry using the software AIDA (Image Data Analyzer v.4.15, Raytest, Isotopenmessgeraete GmbH, Straubenhardt, Germany).

For each PK titration assay on P3s, semilogarithmic curves were obtained by plotting the percentage of protein remaining after digestion (with respect to the undigested sample) against the corresponding PK concentration. The ED₅₀ (e.g. the PK concentration needed to digest 50% of PrP^Sc) for each sample was calculated by means of the equation of the straight line that best fitted the linear portion of the curve (R² ≥ 0.95).

For each sucrose gradient centrifugation assay, the relative amount of each undigested fraction was calculated as a percentage of the sum of all fractions. The aggregation ratio was determined as the ratio between the relative amount of the last six fractions and that of the first six. Finally, the percentage of PrP remaining after digestion in the PK titration curves on THs was not calculated with respect to the undigested sample but rather with respect to the PrP amount detected after digestion at a 0.25 unit/ml PK activity because it was assumed that lower PK dilutions do not completely digest PrP^C.

Statistical Analyses

All statistical analyses were performed with SigmaStat 3.5 (Systat Software Inc., Chicago, IL). One-way analysis of variance (ANOVA) followed by all pairwise multiple comparison procedures were used to detect statistically significant differences among CJD variants in the ED₅₀, aggregation ratio, or PrP^Sc content in grouped fractions of the sucrose gradient assay. Student's t test was used to detect statistically significant differences between MM1 and VV2 in the amount of PrP^Sc in fractions 6, 9, and 12 for each PK dilution used in the titration curve and among the relative amounts of each of the three differently glycosylated PrP^Sc bands after digestion at two PK concentrations for each CJD subtype.

RESULTS

Analysis of PrP Species in Fractions Obtained during PrP^Sc Purification

It is well established that, in both CJD and non-CJD brains, human PrP encompasses truncated fragments in addition to the full-length protein. The known truncated species associated with the non-CJD brain include a major 18-kDa C-terminal fragment, known as C1, which is generated by the cleavage of full-length PrP^C between residues 111 and 112, and a minor ∼20-kDa C-terminal fragment (CTF) (38).

Besides the C1 and the ∼20-kDa CTF, the CJD brain accumulates variable amounts of the PK-resistant and detergent-insoluble core of PrP^Sc, the so-called PrP27–30 (also known as C2), which is generated in vivo by the preferential cleavage of the protein at either residue 82 (type 1, 21 kDa) or 97 (type 2, 19 kDa) (39). Moreover, PK-resistant CTFs of about 12–13 kDa are seen, particularly in the disease variants associated with PrP^Sc type 1 (40, 41).

In the present study, we have assessed PrP^Sc protease resistance and aggregation state across the spectrum of human prions using purified PrP^Sc preparations. The critical separation of PrP^C from PrP^Sc has been obtained through cycles of ultracentrifugation in the presence of detergents, taking advantage of their divergent solubility. To monitor the efficiency of separation of the two PrP species during the purification protocol, we have measured the relative content of the above mentioned truncated fragments in most of the fractions (S1, S2, and P3) obtained. S3 always contained a barely detectable PrP signal, indicating that a complete separation of PrP^C from PrP^Sc in the supernatant is already achieved after the second ultracentrifugation step (S2).

To monitor the degree of PrP^Sc enrichment in the pellet fractions during the purification procedure, we measured the relative amount of truncated PrP fragments in S1 and S2. In control brains, S1 and S2 showed a very similar C1/20-kDa CTF ratio (C1 comprised 66.4–86.0% of the truncated PrP species in S1 and 69.5–84.1% in S2) and a similar overall amount of both full-length and C1 PrP^C, further indicating that virtually all PrP^C is recovered in the S2 fraction (data not shown). At variance with control brains, the S1 fraction from sCJD brains revealed a more significant variability of the relative percentage of C1 (from 34.6 to 74.5% of all truncated species) due to the presence of variable amounts of C2. In the S2, however, the C1/C2 ratio fell within the range of the C1/20-kDa CTF ratio of control brains (C1 ranged from 67.9 to 83.6%), thus excluding the presence of a significant amount of PrP^Sc in S2. The sucrose gradient centrifugation assay of the S2 fraction more specifically showed that virtually all sPrP^Sc is recovered in P2 (Fig. 1). In particular, all PrP^Sc-associated truncated fragments (i.e. the 19- or 21-kDa PrP^Sc cores and the 13-kDa CTF) appeared from the fifth fraction forward, whereas the C1 fragment was the only truncated PrP species in the first four fractions. Thus, because sPrP^Sc (as it is defined in the present work) is expected to sediment in fractions 1–4 (see below), the result makes it very hard to hypothesize a quantitatively relevant sPrP^Sc retention in S2.

FIGURE 1.

PrP truncated species in fractionated S2 samples after sucrose gradient centrifugation. In both MM1 (A) and VV2 (B) sCJD cases, the C1 fragment is clearly detected until the fifth fraction, whereas the 21- and 13-kDa (A) or the 19-kDa (B) PrP^Sc fragments are seen from the fifth fraction onward (arrowheads). Fractions 7–12 have been concentrated 4 times before loading with respect to fractions 1–6. Membranes were probed with mAb SAF 60. Approximate molecular masses are in kDa. CTRL, control.

PrP^Sc Associated with Distinct Human Prion Strains Shows Variable Resistance to Protease Digestion

PK titration curves for each sCJD type, VPSPr, and vCJD performed on both purified PrP^Sc (P3) and THs are shown in Figs. 2, 3, and 4. In P3, the calculated PrP^Sc ED₅₀ (Table 1) was lowest for VV1 and VPSPr and highest for MV 2K/VV2 and vCJD with MM1, MM 2T, and MM 2C showing intermediate values. A similar trend across the spectrum of human prion strains was also obtained by calculating the percentage of detectable PrP^Sc signal at the highest tested PK activity rather than the ED₅₀ (Table 2). Results obtained with THs were highly consistent with those obtained with P3s with the only exception being MM 2T-PrP^Sc, which was slightly more PK-sensitive than MM1-PrP^Sc in the THs (Fig. 4), whereas the opposite was true in P3. Overall, the type 1-linked variants were less PK-resistant than those associated with type 2, although differences were also observed within groups associated with the same PrP^Sc type.

FIGURE 2.

Analyses of protease resistance of purified PrP^Sc by PK titration assay. Purified PrP^Sc from the six sCJD types, VPSPr, and vCJD was run either untreated (first lane) or after digestion with increasing amounts of PK for 1 or 2 h (last lane only) at 37 °C. Membranes were probed with mAb SAF 60. Approximate molecular masses are in kilodaltons.

FIGURE 3.

PK digestion profiles of purified PrP^Sc. The plots represent the amount (mean ± S.D. (error bars)) of PrP^Sc detected after PK digestion expressed as a percentage of the undigested sample for each sCJD group, VPSPr, and vCJD. The PK concentrations used (x axis) are on a logarithmic scale.

FIGURE 4.

PK digestion profiles of total PrP. The plots represent the amount (mean ± S.D. (error bars)) of PrP detected after PK digestion expressed as a percentage of the sample digested with 0.25 unit/ml PK for each sCJD group, VPSPr, and vCJD. The PK concentrations used (x axis) are on a logarithmic scale.

TABLE 1.

ED₅₀ in PK titration assays of PrP^Sc purified from each CJD type tested

ED₅₀ is expressed as mean ± S.D. Statistical significance was evaluated by means of Kruskal-Wallis one-way ANOVA on ranks followed by pairwise multiple comparisons (Q value estimated with Dunn's method; Q critical value = 3.124 for p < 0.05). Statistically significant comparisons included vCJD versus VV1 (Q = 3.811), VV2 versus VV1 (Q = 4.346), VV2 versus MM1 (Q = 3.309), and MV 2K versus VV1 (Q = 3.642).

CJD type	Sample size (n)	ED50
		units/ml
VV1	6	0.034 ± 0.028
VPSPr	2	0.069 ± 0.010
MM1	6	0.093 ± 0.068
MM 2T	4	0.134 ± 0.089
MM 2C	6	0.276 ± 0.210
MV 2K	7	1.407 ± 0.892
VV2	6	4.137 ± 3.562
vCJD	3	5.192 ± 2.378

TABLE 2.

PK dilution values corresponding to the lowest detectable purified PrP^Sc signal on Western blot

Values are expressed as mean ± S.D. and were calculated as the percentage of PrP^Sc remaining after digestion with respect to the undigested sample. For each PK dilution, digestion was carried out for 1 h at 37 °C unless otherwise specified.

CJD type	PK dilution
	5 units/ml	10 units/ml	20 units/ml	20 units/ml, 2 h
	%
VPSPr	9.01 ± 2.24
VV1	4.40 ± 1.71
MM1	8.17 ± 4.65
MM 2T	18.16 ±3.34	9.39 ± 4.79	5.45 ± 4.58
MM 2C	9.62 ± 5.56	4.56 ± 3.75	2.33 ± 1.69
MV 2K			21.95 ± 5.75	10.04 ± 3.57
VV2			27.87 ± 11.58	13.75 ± 3.93
vCJD			30.33 ± 0.85	17.56 ± 0.97

Comparison of the PrP^Sc glycoform ratio obtained after digestion of the protein at different PK concentrations (Table 3) indicated similar kinetics of digestion for each of three glycoforms with only a minor trend toward a preferential digestion of the diglycosylated form, which was most evident in VV1. Interestingly, the PrP^Sc ED₅₀ positively correlated with the relative percentage of diglycosylated PrP^Sc in both “type 1” (VV1 and MM1) and “type 2” (MM 2T, MM 2C, MV 2K, VV2, and vCJD) CJD groups (Table 3). Furthermore, the PrP^Sc ED₅₀ appeared to be inversely correlated with the relative amount of the 13-kDa truncated C-terminal fragment generated by PK digestion (Fig. 2).

TABLE 3.

Effect of PK activity on PrP^Sc glycoform ratio

Values are expressed as relative percentage ± S.D. of diglycosylated (D), monoglycosylated (M), and unglycosylated (U) PrP^Sc based on densitometric analyses of immunoblots probed with mAb 3F4. Statistically significant comparisons (Student's t test or Mann-Whitney rank sum test) included diglycosylated PrP^Sc in VV1 (p < 0.01).

CJD type	Lowest tested PK activitya			PK activity at ED50b
	D	M	U	D	M	U
VV1	21.0 ± 1.6	40.0 ± 4.0	39.0 ± 2.4	17.5 ± 1.0	40.7 ± 3.1	41.8 ± 3.4
MM1	26.1 ± 1.5	49.0 ± 1.6	24.9 ± 2.3	25.9 ± 1.1	47.5 ± 2.4	26.6 ± 1.3
MM 2T	22.0 ± 0.4	44.5 ± 2.3	33.5 ± 1.9	20.3 ± 0.8	44.0 ± 0.5	35.7 ± 0.4
MM 2C	25.4 ± 2.0	42.0 ± 0.5	32.6 ± 1.9	24.6 ± 1.3	43.3 ± 2.4	32.1 ± 2.8
MV 2K	27.9 ± 1.9	40.6 ± 2.0	31.6 ± 0.5	27.5 ± 2.0	39.1 ± 0.8	33.4 ± 1.5
VV2	39.1 ± 5.2	41.9 ± 1.5	19.0 ± 5.1	38.7 ± 3.9	41.2 ± 1.8	20.1 ± 3.5
vCJD	49.1 ± 0.7	34.5 ± 2.3	16.4 ± 1.6	47.0 ± 1.6	35.4 ± 1.9	17.7 ± 0.4

^a Activity was 0.004 unit/ml for VV1 and MM 2T and 0.0315 unit/ml for all other subtypes.

^b Activity was 0.0625 unit/ml for VV1, 0.125 unit/ml for MM1, 0.25 unit/ml for MM 2T and MM 2C, 2 units/ml for MV 2K, 5 units/ml for VV2, and 10 units/ml for vCJD.

In P3, the analyses of the titration curves showed additional differences among disease subtypes. For example, PrP^Sc-MM 2C had a higher ED₅₀ than PrP^Sc-MM 2T (Table 1) despite the latter maintaining about 10% of the signal up to a higher PK concentration (10 versus 5 units/ml) (Table 2). Finally, it is worth noting that the ED₅₀ of PrP^Sc purified from VPSPr carrying the MM or MV genotype was within the range of those observed among the classic sCJD variants.

PrP^Sc Associated with Distinct Human Prion Strains Shows Differences in Aggregation State

The analyses of PrP^Sc distribution among the fractions collected after PrP^Sc purification and sucrose gradient ultracentrifugation showed that PrP^Sc preferentially localizes in the intermediate and late fractions irrespective of the disease subtype (Fig. 5, A–C). Nevertheless, the fraction containing the highest amount of PrP^Sc correlated to some extent with the CJD subtype (i.e. fraction 8 in VPSPr and VV1; fraction 9 in MM1, MV 2K, VV2, and MM 2T; fraction 10 in vCJD; and fraction 12 in MM 2C). Furthermore, the distribution of the largest PrP^Sc aggregates (i.e. fractions 9–12) was significantly influenced by both the type of PrP^Sc and the codon 129 genotype with both type 2 and methionine at codon 129 favoring the distribution of the abnormal protein in the late fractions (Fig. 5D).

FIGURE 5.

PrP^Sc aggregate distribution among fractions after sucrose gradient centrifugation assay. A–C, for each CJD type and VPSPr, the amount (mean ± S.D. (error bars)) of PrP^Sc in each fraction collected after sucrose gradient centrifugation is expressed as a percentage of all fractions. D, the PrP^Sc amount of combined fractions 1–4, 5–8, and 9–12 is shown. Note that the PrP^Sc content of fractions 1–4, containing the least aggregated and fully PK-sensitive PrP^Sc, does not exceed 10% in all groups. Nevertheless, the PrP^Sc content in these slowly sedimenting fractions is significantly higher in MM 2C than in VV2, MV 2K, MM 2T, MM1 (p < 0.001), VV1, and VPSPr (p < 0.002) (one-way ANOVA test followed by all pairwise multiple comparisons with the Holm-Sidak method). Intermediate (5–8) and bottom fractions (9–12) contain partially PK-resistant PrP^Sc aggregates that also vary in relative proportion among CJD types. The PrP^Sc amount in fractions 9–12 was statistically different in the following pairs: (i) VV1 versus vCJD, MM 2C, MM 2T, and MV 2K (p < 0.001); (ii) VPSPr versus vCJD, MM 2T, MM 2C (p < 0.001), MV 2K (p = 0.001), and VV2 (p = 0.002); and (iii) MM1 versus vCJD (p < 0.001) (one-way ANOVA test followed by Holm-Sidak method for all pairwise multiple comparisons).

To obtain a semiquantitative index of the overall state of PrP^Sc aggregation for each CJD type, we calculated the ratio between the amounts of PrP^Sc in fractions 7–12 and those in fractions 1–6. Statistical analyses were significant between groups at the very extreme of the range (Table 4). Thus, VPSPr and VV1, the groups with the lowest aggregation ratio, showed significant differences with respect to those with the highest values such as VV2 and MV 2K or vCJD. In addition, PrP^Sc from MM1 significantly differed from that of vCJD PrP^Sc. Finally, the analyses of the PrP^Sc aggregation ratio also distinguished MM 2C from MM 2T, two rare sCJD variants sharing the PrP primary sequence and many PrP^Sc properties including the fragment size after PK digestion, the degree of protease resistance, and the propensity to mainly form aggregates of relatively large size (i.e. fractions 9–12).

TABLE 4.

PrP^Sc aggregation ratio for each CJD type

The aggregation ratio is expressed as mean ± S.D. Statistical significance was evaluated by means of one-way ANOVA followed by pairwise multiple comparisons carried out by the Holm-Sidak method. Statistically significant comparisons included VPSPr versus MM 2T, versus vCJD, versus VV2 (p < 0.001), and versus MV 2K (p = 0.001); VV1 versus MM 2T, versus vCJD (p < 0.001), and versus VV2 (p = 0.001); MM 2C versus MM 2T and versus vCJD (p < 0.001); and MM1 versus MM 2T and versus vCJD (p < 0.001).

CJD type	Sample size (n)	Aggregation ratio
VPSPr	2	3.76 ± 0.28
VV1	6	5.47 ± 0.46
MM 2C	5	5.68 ± 0.99
MM1	6	6.43 ± 2.15
MV 2K	6	8.38 ± 1.93
VV2	6	8.68 ± 1.22
MM 2T	4	10.92 ± 1.44
vCJD	3	11.65 ± 3.35

To assess the relationship between PrP^Sc aggregation and PK resistance for each strain, we then compared the PrP^Sc “aggregation ratio” with the ED₅₀. The results of this analysis (Tables 1 and 4) showed a positive correlation between the two variables with the only exception being MM 2T-PrP^Sc, which showed an intermediate degree of PK resistance despite the very high aggregation ratio with the latter being comparable with that of vCJD-PrP^Sc, the most aggregated and most PK-resistant PrP^Sc type.

Detection of sPrP^Sc

Digestion of the P3 fractions obtained from the sucrose gradient centrifugation assay revealed a totally PK-sensitive component in each type. Fully PK-sensitive PrP^Sc distributed exclusively among fractions 1–4 (Fig. 6) irrespective of the CJD type, indicating that this sPrP^Sc component is less aggregated than rPrP^Sc. Overall, sPrP^Sc accounted for less than 10% of the whole PrP^Sc with MM 2C showing the highest and VV2 showing the lowest relative amount (Fig. 5D). Thus, the amount of this sPrP^Sc fraction seems to be unrelated to the overall degree of PrP^Sc PK resistance even if it is contributing to the aggregation ratio.

FIGURE 6.

Detection of fully sensitive PrP^Sc in slowly sedimenting fractions. Western blot analysis of PrP^Sc distribution among fractions collected after sucrose gradient centrifugation in MM 2C is shown. A, PrP^Sc distribution among the 12 fractions before PK digestion. Serial dilutions of a sample with known PrP^Sc content have been used as a standard curve. B, the PrP^Sc amount in the same fractions after PK digestion (PK dilution used, 0.25 unit/ml). Note that the signal in fractions 1–4 has completely disappeared, whereas the standard curve shows a comparable intensity of signal. C, overexposure of A showing that in fractions 1–4 (i) PrP dimers, typical of PrP^Sc and absent in PrP^C, are well represented (arrowhead) and (ii) the PrP glycosylation pattern is typical of PrP^Sc and different from that of PrP^C. D, overexposure of B demonstrating the complete disappearance of sPrP^Sc signal after PK digestion. Membranes were probed with mAb SAF 60. Approximate molecular masses are in kilodaltons.

The PK sensitivity of the PrP^Sc recovered in fractions 1–4 was comparable with that of PrP^C because after adjusting for the total protein concentration it was almost completely digested by the minimal PK concentration required for PrP^C digestion (data not shown). Thus, it seems that the aggregate size represents a main determinant of PrP^Sc resistance to PK digestion. Accordingly, changes in rPrP^Sc aggregate size could potentially affect sPrP^Sc content. Indeed, prolonged sonication after the ultracentrifugation steps during PrP^Sc purification resulted in an increase of sPrP^Sc amount in fractions 1–4 (data not shown).

rPrP^Sc PK Resistance in the Same Fraction from Different Types: MM1 Versus VV2

To determine whether CJD type-specific rPrP^Sc preparations with similar aggregation size maintain or not their difference in PK resistance, we performed PK titration curves on selected fractions of the sucrose gradient assay. For this experiment, we chose to compare fractions 6, 9, and 12 between MM1 and VV2, two common human sCJD variants, which showed statistically significant differences in their PK resistance (e.g. ED₅₀) but not in their aggregation ratio (Fig. 7). By showing that VV2-rPrP^Sc is significantly more resistant than MM1-rPrP^Sc in each of the fractions analyzed, the results replicated the differences observed for the PK titration curves of the whole purified PrP^Sc (Fig. 8), indicating that this fundamental property of prions is also determined by structural PrP^Sc properties that are independent from the relative size of the protein aggregates.

FIGURE 7.

Analysis of protease resistance of purified PrP^Sc fractions with similar aggregation size: comparison between MM1 and VV2. For each sample, PK titration curves have been performed on fractions 6, 9, and 12 collected after subjecting purified PrP^Sc to sucrose gradient centrifugation. Samples were run either untreated (first lane) or after digestion with increasing amounts of PK for 1 or 2 h (last lane only) at 37 °C. Membranes were probed with mAb SAF 60. Approximate molecular masses are in kilodaltons.

FIGURE 8.

PK digestion profiles of purified PrP^Sc fractions with similar aggregation size: comparison between MM1 and VV2. The plots represent the amount (mean ± S.D. (error bars)) of PrP^Sc detected after PK digestion in fractions 6, 9, and 12 expressed as a percentage of the undigested sample. The PK concentrations used (x axis) are in a logarithmic scale. An asterisk (*) indicates statistically significant differences between MM1 and VV2 (Student's t test or Mann-Whitney rank sum test).

DISCUSSION

In the present study, we have carried out the first systematic analysis of PrP^Sc protease resistance and aggregation state across the spectrum of human sporadic and vCJD prions. The results show significant strain-specific differences in the PK digestion profile of human PrP^Sc that are only partially explained by differences in aggregate size, indicating that other factors also determine this fundamental property of prions.

Overall, CJD variants linked to PrP^Sc type 1 (VV1 and MM1 subtypes) were less PK-resistant than those associated with PrP^Sc type 2 (MM 2T, MM 2C, MV 2K, VV2, and vCJD), although clear differences were also seen among CJD prions sharing the same PrP^Sc type. In particular, VV1 PrP^Sc was by far the least resistant, whereas MV 2K/VV2- and vCJD-associated PrP^Sc showed the highest resistance to PK digestion. Interestingly, the PrP^Sc ED₅₀ also appeared to be inversely correlated with the relative amount of the 13-kDa truncated C-terminal fragment generated by PK digestion (41), suggesting that the protease resistance of PrP^Sc also depends on the accessibility of PK to the PrP^Sc C terminus.

By showing that purified PrP^Sc forming low molecular mass aggregates is digested completely at a PK activity comparable with the lowest activity that is needed to fully digest PrP^C, our data confirm that a minimal aggregation size for the PrP^Sc molecules is required to make the protein resistant to proteolysis and that this property, at least partially, depends on its quaternary structure. Furthermore, our data confirm the existence of a bona fide sPrP^Sc across the whole spectrum of human prions. However, our semiquantitative analyses clearly show that this slowly sedimenting sPrP^Sc represents only a relatively minor component of abnormal PrP not exceeding 10% of total detergent-insoluble PrP^Sc in any of the human sporadic TSE subtypes. Thus, previous concerns raised about the common practice of diagnosing human prion diseases by means of rPrP^Sc detection and characterization (24) should be, at least for classic sCJD and vCJD, significantly reduced. Moreover, our finding of a comparable relative amount of sPrP^Sc across the whole spectrum of CJD prions comprising six distinct strains an including VPSPr linked to MM or MV at codon 129 argues against the idea that sPrP^Sc, or at least its slowly sedimenting component, plays a major role in prion strain determination in human sporadic prion diseases.

The significant difference between our estimate of sPrP^Sc among sCJD isolates and that of Safar et al. (24) may depend on methodological aspects or data interpretation. First, the two approaches differ in the means by which they distinguish between PrP^C and PrP^Sc. Indeed, although our method completely separated PrP^C in a verifiable manner (Ref. 26 and present study), CDI maintains PrP^C in the sample and estimates the amount of sPrP^Sc assuming that the epitope unmasking induced by guanidine hydrochloride is specific for PrP^Sc and as a consequence that PrP^C would not contribute to the increase of the PrP signal after denaturation.

Second, although our estimate of sPrP^Sc has been obtained after the physical separation of a fully PK-sensitive PrP^Sc fraction forming aggregates of relatively low size, CDI measures sPrP^Sc as a part of the whole PrP^Sc without defining any actual structural or physical difference between the two (s versus r) PrP^Sc species, raising the issue of whether the sPrP^Sc signal detected by CDI reflects the properties of a distinct PrP^Sc molecule, is an indivisible part of the features of the PrP^Sc molecule, or a mixture of both.

Furthermore, it is noteworthy that PrP^Sc was originally defined as an abnormal conformer that is only partially resistant to protease digestion, a property that is also well illustrated in the present study. Thus, the CDI estimate of the amount of sPrP^Sc may also comprise this phenomenon. However, it is well known that the experimental conditions in which the protease digestion is performed (i.e. pH, temperature, and protein concentration) may dramatically vary the resistance of a given substrate (36). In addition, there are significant differences in the degree of protease resistance of PrP^Sc that depend on the type of prion isolate as shown in the present and a previous study from our group (42) for human PrP^Sc. Altogether, these considerations underline the difficulty and limits of creating a distinction between PrP^Sc isoforms (s versus r as it is increasingly reported in the literature) only based on a single digestion condition and without any definition of the actual structural or physical differences between the two PrP species. Furthermore, the strict dependence on the experimental conditions used would make any interlaboratory quantitative comparison between the putative “sensitive” and resistant components extremely difficult.

Our detailed PK digestion curves on the whole purified PrP^Sc, by showing the progression of the loss of PrP^Sc signal at increasing PK activity for each of the sCJD prions and vCJD, exemplify some of the issues discussed above and show the heterogeneity of this additional potential “sPrP^Sc-like” component according to the CJD subtype at a given PK activity. When the percentage of PrP^Sc signal loss is considered at the lowest PK activity we have used for the most resistant strains, which was still capable of trimming the N-terminal portion of PrP^Sc, generating PrP27–30, it was 1.9% in vCJD, 22.3% in VV2, and 53.4% in VV1. Nevertheless, it is noteworthy that even in the most sensitive prion isolate tested here, the VV1, the disappearance of the large majority of the PrP^Sc signal (85%) was only obtained in stringent experimental conditions (PK activity of 200 μg/ml, pH 7.4 and a very low protein concentration of 150 μg/ml). Therefore, it appears very unlikely that in less stringent experimental conditions such as those used in the CDI the loss of PrP^Sc after PK digestion would reach the amount estimated by CDI (24) in any of the CJD subtypes.

To further examine the relationship between aggregation size and PK sensitivity, we calculated the aggregation ratio, which measures the proportion of smaller PrP aggregates with respect to larger aggregates. We found a trend of values among the CJD strains reproducing, to some extent, those obtained with the PK digestion profiles. In particular, there was a positive correlation between the aggregation ratio and both PrP^Sc type 2 and codon 129 MM genotype with the vCJD group showing the highest value. Similarly, the relative amount of PrP^Sc among the fastest sedimenting fractions (9–12) also showed a statistically significant increase for both Met polymorphism and type 2, although in this case the value was highest for MM 2C rather than vCJD. Overall, these data are in agreement with the results of the study of Kobayashi et al. (43) suggesting that sCJD MM2 brains contain larger PrP^Sc aggregates than MM1 brains and with the work of Pham et al. (44) demonstrating that human recombinant PrP is more prone to aggregate when carrying Met at codon 129 instead of Val.

According to our findings, however, the positive correlation between the aggregation ratio and the ED₅₀ expressing the resistance to proteolysis is not complete given, for example, that MM1 cases were significantly different from VV2s in their PK resistance despite the similar aggregation ratio. This is also in keeping with a previous observation obtained in Gerstmann-Sträussler-Scheinker disease or atypical scrapie where PrP^Sc forms relatively large aggregates that are easily digested by PK (26, 28–30, 45). To further address this issue, we also compared the PK resistance of single fractions containing aggregates of similar sizes obtained from different CJD strains. By showing that VV2 prions remain more PK-resistant than MM1 prions even when the influence of the size of the aggregates is eliminated, our findings strongly suggest that PrP^Sc sensitivity is also caused by other strain-specific factors. Given that this strain-specific difference in PK sensitivity becomes significant only when the PK activity is relatively high and that PrP^Sc is completely digested under a minimal aggregation size, indicating that PrP^Sc aggregation is a necessary condition for PK resistance to occur, we propose that this second factor comprises the stability of PrP^Sc aggregates. In other words, the most PK-resistant prions would be characterized not only by PrP^Sc aggregates of larger size but also kept together by stronger interactions. According to Masel and Jansen (46), PK digestion is biphasic: during the first phase, PK would attack single monomers (or exposed sites of aggregated polymers), whereas during the second phase, polymers must disaggregate to be digested. As a consequence, in this latter phase the digestion rate would exclusively depend on the rate of PrP^Sc disaggregation. Thus, if PrP^Sc associated with different CJD subtypes has indeed a heterogeneous propensity to fragmentation, then PK digestion will occur at different rates even in equally aggregated samples. This scenario (Fig. 9) would not only fit with our findings but also accommodate previous observations, particularly in Gerstmann-Sträussler-Scheinker disease or atypical scrapie where PrP^Sc forms relatively large aggregates that are easily digested by PK (26, 28–30, 35, 45). These could instead be explained by postulating that PrP^Sc forms less stable aggregates compared with classic CJD or scrapie rather than smaller aggregates. It is noteworthy that differences in aggregate frangibility among strains have already been demonstrated for yeast prions (47–49).

FIGURE 9.

PrP^Sc aggregate stability: a model to explain the heterogeneity of PrP^Sc resistance to proteolysis among prion strains. While strain A shows a compact PrP^Sc core with low tendency to crumble into monomers, strain B is characterized by a more unstable core that is more prone to break up into separate monomers. In the presence of a relatively low PK activity, both strains A and B produce a few monomers that are easily digested together with the exposed portions of the whole core. In the presence of a relatively higher PK activity, the fragmentation into new monomers is significantly accelerated only for the PrP^Sc core of strain B, whereas the more compact PrP^Sc core of strain A is less affected.

Interestingly, this scenario would implicate that PrP^Sc resistance to protease digestion could be reduced by lowering its aggregation size, although proportionally to the strength that keeps the PrP^Sc particles together. Previous studies have demonstrated that sonication reduces PrP^Sc aggregate size (50–52), and our preliminary studies suggest that a brief sonication cycle reduces PrP^Sc resistance to digestion in VV1 and MM1 subtypes but not in MV 2K or VV2s. The reduction in size of PrP^Sc aggregates would likely make them more susceptible to endogenous protease activity and may increase their clearance, which would have a significant therapeutic implication. To this aim, it would be important to understand the factors modulating PrP^Sc aggregate stability. Given that the two sCJD subgroups with the most significant difference in aggregate stability (VV1 and VV2) have identical PRNP sequence, it is likely that factors other than primary PrP sequence are involved. Several studies have pointed to the glycosaminoglycans as major players in protein aggregation related to neurodegeneration (53) and as prion disease therapeutic targets as well (54). Increasing evidence suggests that carbohydrate-involving interactions play a role in protein aggregation and aggregate stabilization. These would be promoted, for example, by glycation related to oxidative stress, which occurs between lateral amino acidic chains of the protein and carbonyl-containing groups, causing the formation of cross-links that stabilize protein aggregates (55). PrP contains a glycosaminoglycan-binding motif at its N terminus (56) and can be glycated in the same region (57). Furthermore, PrP contains two glycosylation sites (Asn-181 and Asn-197) that can carry N-linked oligosaccharides. The nature of these carbohydrates is still poorly understood, and it is not clear whether they are qualitatively different among sCJD subtypes. Nevertheless, host PrP glycosylation patterns can affect disease progression, alter the infectious properties of a specific prion strain, and even protect against disease onset (58–60). Our finding of a positive correlation between the relative percentage of diglycosylated PrP^Sc and the overall degree of PrP^Sc resistance to PK digestion also supports the notion that N-linked glycans influence protein aggregate stability. On the other hand, the observation that the kinetics of digestion of the three PrP^Sc glycoforms is fairly similar is only in apparent contrast with the idea formulated above because evidence indicates that PrP^Sc particles or aggregates always contain a mixture of glycoforms (61). Thus, the possibility that N-linked oligosaccharides modulate stability by themselves or contribute together with other protein-linked carbohydrate structures to determine the strain-specific PrP^Sc aggregate stability remains a scenario that should be considered and further explored.

In summary, in the present study we have documented that human prion strains differ in PrP^Sc PK sensitivity over a 100-fold range of PK concentration and that the differences stem from both aggregate stability and size. By providing new insight into prion strain variability with respect to PrP^Sc PK sensitivity and aggregation state, our findings could also serve as a starting point for understanding the properties of other disease-associated misfolded proteins. Indeed, increasing evidence argues that other protein aggregates (i.e. tau, α-synuclein, amyloid-β, and huntingtin aggregates), like prions, can seed the misfolding of their normal conformers (62) and even show a “strain” specificity in their PK digestion profile and in vivo cross-seeding ability as was recently shown for α-synuclein (63).