Gerstmann-Sträussler-Scheinker Disease and “Anchorless Prion Protein” Mice Share Prion Conformational Properties Diverging from Sporadic Creutzfeldt-Jakob Disease^*

Synthetic Peptides and Antibodies

Human synthetic PrP peptides spanning sequences 23–230, 90–230, 105–230, and 121–230 were purchased (Alicon AG, Zurich, CH); PrP peptide 82–146 was kindly donated by Dr. M. Salmona. The following mouse monoclonal antibodies recognizing different human PrP epitopes were used: 3F4, residues 108–111 (Signet Laboratories), 6D11, residues 93–109 (Signet Laboratories), ICSM-35, residues 93–102 (d-Gen UK), 12B2, residues 89–93 (kindly donated by Dr. J.P.M. Langeveld), SAF70, residues 142–160 (Cayman Chemicals), 6H4, residues 144–152 (Prionics, CH), 4G11, residues 199–216, and 3E2, residues 214–231 (kindly donated by Dr. L. Capucci).

Animal Inoculation

All mice were housed at the Rocky Mountain Laboratories (RML) in an AAALAC-accredited facility. Research protocols and experimentation were approved by the NIH RML Animal Care and Use Committee. Transgenic GPI anchorless PrP mice (tg44+/+) were generated as previously described (13). Four to six-week-old mice were inoculated intracerebrally with 50 μl of a 1% brain homogenate of RML scrapie containing 0.7–1.0 × 10⁶ ID₅₀. One ID₅₀ is the dose causing infection in 50% of C57BL/10SnJ mice. Animals were observed daily for onset and progression of scrapie. Mice were euthanized when clinical signs were consistent and progressive.

sCJD Tissue Samples

Brain samples were obtained from 29 cases of definite sCJD, 11 methionine homozygous at codon 129 with type 1 PrP^Sc (MM1), 3 methionine/valine with type 1 PrP^Sc (MV1), 3 MM with type 2 PrP^Sc (MM2), 7 MV2, 5 VV2, and 1 case of variant CJD (vCJD), diagnosed according to current criteria (17). Post-mortem intervals ranged from 4 to 30 h. Genomic DNA, extracted from frozen brain tissues, was searched for PRNP mutations and M/V polymorphism at codon 129. Neuropathological and immunohistochemical studies were performed as described previously (18).

Immunoblot Analysis

Brain samples were homogenized in 9 volumes of lysis buffer (100 mm sodium chloride, 10 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mm Tris, pH 7.4). Aliquots were adjusted to a final concentration of 50 μg of proteinase K (Roche Applied Science, Germany) per milliliter and incubated at 37 °C for 60 min; protease digestion was quenched by adding PMSF to a final concentration of 2 mm (Boehringer, Manheim). In some experiments, PMFS was omitted and samples were dissolved in Laemmli buffer (3% SDS, 3% β-mercaptoethanol, 2 mm EDTA, 10% glycerol, 62.5 mm Tris, pH 6.8), before boiling at 100 °C for 5 min. The combined exposure of samples to the denaturing anionic detergent SDS and PK, was exploited to solubilize non-amyloid PrP complexes and assess the conformation of PrP^Sc species under conditions altering the quaternary and tertiary structure of PrP^Sc.

For N-deglycosylation, samples were treated with N-glycosidase F (PNGase-F) according to the manufacturer's instruction (Roche Applied Science) for 8 h at 37 °C. Samples were dissolved in Laemmli buffer and boiled for 5 min. An equivalent of 0.4 mg of wet tissue was loaded on 13% SDS-PAGE gels and proteins were transferred onto PVDF membrane (Immobilon P, Millipore) for 2 h at 60 V. Membranes were blocked with 1% nonfat dry milk in TBST (10 mm Tris, 150 mm NaCl, 0.1% Tween-20, pH 7.5) for 1 h at 37 °C and incubated overnight at 4 °C with anti-human PrP monoclonal antibodies (3F4, 1:5,000; 6D11, 1:3,000; ICSM-35, 1:1,000; 12B2, 1:8.000; SAF70, 1:1,000; 6H4, 1:5,000; 4G11, 1:1,000; 3E2, 1: 3,000). Blots were developed with an enhanced chemiluminescence system (ECL, Amersham Biosciences) and PrP visualized on autoradiographic films (Hyperfilm, Amersham Biosciences). Films were scanned by using a densitometer (GS-200, Bio-Rad).

Two-Dimensional Gel Electrophoresis (2D-PAGE)

For isoelectric focusing (IEF), using immobilized pH gradients (IPG) in the first dimension, pre-cast gels with a linear pH range of 3–10 were used (Bio-Rad). Before IEF, the dry gels were reswollen for 14–15 h in 125 μl of buffer (6 m urea, 2 m thiourea, 5% β-mercaptoethanol, 2% Nonidet P-40, and 2% Ampholytes) containing the equivalent of 2 mg of wet tissue. IEF was carried out at 20 °C for 4 h with raising voltage (500–8000 V), in a cooled horizontal electrophoresis unit (IPGphor, Pharmacia). For the second dimension, the IPG strips were equilibrated for 20 min in 50 mm Tris-HCl, 6 m urea, 10% glycerol, 2% SDS, and a trace of bromphenol blue and loaded on a 16% SDS-PAGE (19). Immunoblotting was performed as described above.

Molecular Typing of Anchorless and Wild Type Prions

Immunoblots with 6D11 of non-PK-treated brain homogenates (BHs) from “anchorless PrP” Tg mice and “anchored PrP” C57BL mice, both infected with the RML scrapie strain, showed that anchorless PrP resolved in 2 bands (Fig. 1A, lane 2), representing mono- and unglycosylated forms, as opposed to the customary separation of anchored PrP under di-, mono- and unglycosylated species (Fig. 1A, lane 3). As expected, the unglycosylated anchorless PrP band co-migrated with the PrP²³⁻²³⁰ synthetic peptide at 23 kDa (Fig. 1A, lanes 1 and 2). Higher molecular mass forms, consistent with PrP multimers, in addition to a barely detectable 16 kDa truncated fragment, were observed in “anchorless PrP” Tg mice; conversely, anchored PrP showed an additional C-terminal truncated fragment migrating in a 19-kDa zone, accounting for the C2 fragment, or the unglycosylated fragment generated by endogenous proteolysis (5). Following treatment with PK, anchorless PrP^Sc separated under mono- and unglycosylated isoforms, migrating at ∼ 21 and 16 kDa (Fig. 1A, lane 4), whereas anchored PrP^Sc glycoforms migrated at ∼28, 24, and 19 kDa (Fig. 1A, lane 5). Hence, the unglycosylated fragment of anchorless PrP^Sc migrated ∼3 kDa faster than the corresponding fragment of anchored PrP^Sc, in keeping with the estimated molecular mass of the GPI anchor. Enzymatic deglycosylation of anchorless PrP yielded a 23-kDa band, in addition to a barely visible 16 kDa band, and reduced anchored PrP to two distinct bands of 26 and 19 kDa, representing full-length PrP and the C2 fragment (Fig. 1A, lanes 6 and 7); further treatment with PK generated a core fragment of 16 kDa for anchorless PrP and 19 kDa for anchored PrP (Fig. 1A, lanes 8 and 9). Membranes probed with mAb SAF70 overlapped results observed with 6D11 (Fig. 1B, lanes 2–9), with the exception of an additional lower band detected in wild type mice, corresponding to the C1 fragment (Fig. 1B, lane 7). Taken together, the foregoing results were consistent with the GPI anchor contributing for ∼3 kDa to the orthogonal migration of PrP, either before or after PK proteolysis.

Two-Dimensional Mapping of Anchorless and Wild Type Prions

By 2D-PAGE, we next compared the isoelectric point (reflecting the net charge) and the mobility (determined by the molecular mass) of anchorless PrP and anchored PrP glycoforms. Reference SDS-PAGE and 2D-PAGE maps of human synthetic PrP peptides were as follows: the 23–230 peptide migrated at 23 kDa, pI 9.4, the 90–230 at 16 kDa, pI 7.9, and the 105–230 at 14.5 kDa, pI 6.6 (Fig. 2A, panels a, b, and table).

Immunoblots with 6D11 (data not shown) and SAF70 of BHs from anchorless PrP Tg mice showed that the unglycosylated PrP isoform migrated at 23 kDa, pI 9.4, matching the orthogonal and horizontal mobilities of the synthetic PrP peptide 23–230, whereas monoglycosylated isoforms showed a string pattern in a 28-kDa zone, pI 8–9.2 (Fig. 2B, panels a and b), all reduced to a single 23-kDa spot upon enzymatic deglycosylation (Fig. 2B, panels c and d). In overexposed films a 16-kDa spot, matching the migration of the 90–230 peptide, was seen.

Immunoblots with 6D11 of wt mice BHs showed that PrP separated as trains of spots migrating between 35 and 19kDa, pIs ∼4–8.5, including glycosylated and unglycosylated isoforms of the full-length PrP and glycoforms of the C2 fragment (data not shown); upon deglycosylation, PrP isoforms resumed to two main 26-kDa and 19-kDa spots, pIs 8.4 and 7.2–8.2, accounting for the unglycosylated full-length PrP and the C2 fragment. Membranes probed with SAF70, which preferentially binds glycosylated forms of C1, disclosed a PrP pattern dominated by a set of spots with an acidic migration (Fig. 2B, panels e and f), all reduced to three set of spots after deglycosylation, including full-length PrP, C2, and C1 fragments (Fig. 2B, panels g and h). Notably, intra-sample differences in PrP decoration, observed between SDS-PAGE and 2D-PAGE, reflect the facilitation or restriction of mAbs binding to PrP molecules denatured by alternate protocols.

Taken together, using a high sensitive proteomic approach we confirmed that lack of the GPI anchor induces an alkaline shift of ∼1 pH unit in the horizontal migration of PrP, and also that anchorless PrP is characterized by immature glycosylation and altered endogenous proteolytic processing (13).

After PK digestion, anchorless PrP^Sc separated into minor glycosylated spots in a 19-kDa zone, in addition to well-represented unglycosylated 16 kDa isoforms, consistent with multiple ragged N-terminal ends of the unglycosylated PrP27–30 fragment (Fig. 2C, panels a and b), all reduced to a single train of 16-kDa spots, pIs 7.5–8, after PNGase treatment (Fig. 2C, panels c and d).

As expected, immunoblots with SAF70 of PK-digested BHs from C57BL mice showed the typical separation of PrP^Sc in three series of variably glycosylated isoforms (panels e and f), all resumed to three contiguous 19-kDa spots upon deglycosylation (Fig. 2C, panels e and f). After deglycosylation C-terminal truncated fragments (CTFs), migrating at 14–16 kDa, pI range 4–6, were observed (Fig. 2C, panels g and h). Taken together, besides differences in migration, anchorless PrP^Sc does not generate CTFs under protease treatment.

Molecular Characteristics of PrP^Sc in sCJD Subtypes and vCJD

In previous 2D-PAGE studies of PrP^Sc in different sCJD subtypes and vCJD, we were unable to detect molecular forms consistent with anchorless PrP species. Here, we first assessed the SDS-PAGE migration of type-1 and type-2 PrP^Sc, as compared with the map of human synthetic PrP peptides. Accordingly, the unglycosylated PrP27–30 fragment migrates at 19.5-kDa in type-1 PrP^Sc, and 17-kDa in type-2 PrP^Sc (Fig. 3, A and B), which is at variance with their conventionally reported migration at 21 and 19 kDa (20). Next, we reassessed the 2D-PAGE migration of the core fragment in different sCJD subtypes using anti-PrP mAbs 3F4 and 3E2. In sCJD MM1, the PrP27–30 core fragment migrates as a single tailed 19.5-kDa spot centered at pI 7.0, whereas in sCJD MM2 the core fragment separated as three major 17.5-kDa spots, pI range 6.0–7.0, as an effect of multiple cleavage sites (Fig. 3C). Differences in migration between PrP^Sc of MM1 and MM2 subtypes were better visualized in a sCJD case with co-occurrence of both PrP types (MM1 + 2), where the core fragment of type-1 PrP^Sc and the most basic spot of the type-2 PrP^Sc train shared a spot at pI 7.0, with diverging molecular masses. In contrast to sCJD MM1, in MV2 and VV2 subtypes PrP^Sc separated as multiple spots in a wider pI range, spanning from 6.6 to 8.0, indicating a larger heterogeneity of PrP N-terminal variants, whereas in vCJD, PrP^Sc resolved in three major spots (18, 21).

By probing membranes with mAb 3E2 (epitope 214–231), the 2D-patterns of PrP^Sc core fragments in different sCJD subtypes and in vCJD overlapped those observed with 3F4; additionally, acidic C-terminal truncated fragments were seen in distinct sCJD subtypes, as already reported (Fig. 3D). Taken together, in MM1, MM2, and MM1 + 2 sCJD subtypes, each single spot of the core fragment is 1.0 pH unit more acidic than the corresponding synthetic peptide and ∼2.5 kDa slower, as expected for anchored PrP species. At variance, MV2, VV2, and vCJD show the presence of additional basic spots with migration between 7.0 and 8.0. Although caution is needed in assigning these species to anchorless PrP forms, the lack of the expected 2.5 kDa shift in their orthogonal migration and the recognition by mAb3E2 rule out the hypothesis that they could represent anchorless PrP species, rather suggesting the presence of molecular forms with modified GPI composition.

Antibody Mapping of PrP^Sc in sCJD and vCJD: No Evidence for Anchorless Species

Previously, Notari et al. reported on the constitutive expression of anchorless PrP^Sc species in all sCJD subtypes, and their enrichment after PK digestion (16). Anchorless PrP quasispecies were tentatively identified based on their migration, ∼2 kDa faster than the core fragment, and their detection with mAbs directed to epitopes along PrP residues 89–221, including 12B2 and 3F4, but not with a rabbit antiserum directed to residues 220–231.

In the present study, PrP^Sc characterization in different sCJD subtypes and vCJD, did not show additional bands, consistent with anchorless PrP species, either before (Fig. 4A, panels a and b) or after PK treatment (Fig. 4A, panels c and d). In particular, immunoblots with anti-PrP mAbs 6D11 (epitope 93–109) and ICSM-35 (epitope 93–102) showed only a different affinity and variability of the PrP^Sc glycosylation profile, but no additional faster migrating bands (data not shown). At variance, immunoblots with 12B2, recognizing an epitope at position 89–93 of human PrP, showed that the unglycosylated PrP27–30 fragment co-migrated in a 19.5 kDa zone in all sCJD subtypes, either with type-1 or type-2 PrP^Sc and vCJD. An exception to this pattern was the lack of recognition of PrP27–30 in CJD MM2 subtype, and the detection of a PrP band migrating in a 17 kDa zone in four out seven sCJD MV2 cases (Fig. 4B, panels a and b). These findings further support previous demonstration that type-1 PrP^Sc is variably expressed in vCJD and sCJD cases with type-2 PrP^Sc, either homozygous or heterozygous at codon 129. The lack of PrP27–30 staining in MM2 cases is consistent with this molecular subtype being mostly composed by the N-terminal variant of PrP27–30 starting at S97, hence lacking the 12B2 epitope (21).

After 2D-PAGE separation, immunoblots with 12B2 confirmed the co-expression of type-1 and type-2 unglycosylated PrP27–30 spots in four MV2 cases. As expected, these spots shared a similar horizontal migration, within a 6.0–7.0 pI range, but differed in their orthogonal mobility at 19.5 and 17 kDa (Fig. 4C). As compared with the pattern obtained with mAbs 3E2 and 3F4 (see Fig. 3, C and D), in MV2 cases the reduced number of spots, and in particular the absence of basic 17.5-kDa isoforms, is consistent with mAb 12B2 (epitope 89–93) missing the capture of G92, S97, and W99 N-terminal species that are highly expressed in this sCJD subtype (21). Therefore, using an extensive panel of anti-PrP mAbs and a high sensitive separation technique, we were unable to confirm the presence of anchorless PrP species, as suggested by Notari et al. using a conventional SDS-PAGE technique and an anti-PrP rabbit antiserum (16).

GSS-like Internal PrP^Sc Fragments Are Generated in “Anchorless PrP” Tg Mice, but Not in sCJD and vCJD, following PK Digestion under Inactivating Conditions

We next investigated the influence of GPI anchor in PrP^Sc conformation under conditions where PMSF was omitted. Using this protocol, PK-digested BHs from anchorless PrP Tg mice, showed, in addition to PrP27–30, bands migrating at 14 and 8 kDa, both recognized by mAbs binding to PrP residues 89–109, including 12B2, 6D11, and ICSM-35 (Fig. 5A). Conversely, mAb 6H4 faintly stained the 14-kDa band, and 4G11 detected barely visible bands in a higher molecular zone, corresponding to previously identified C-terminal fragments (Fig. 5A). At variance, the 14 and 8-kDa bands were not observed in anchored PrP C57BL mice. Interestingly, omission of PMSF induced a reduction of large PrP aggregates, migrating in a high zone, suggesting that these aggregates originate the PrP27–30 in wild type mice and internal fragments in “anchorless PrP” mice, either at 1 or 4 h of PK digestion (Fig. 5B). Under the same experimental conditions, no truncated fragments migrating in the 14–8 kDa range were observed in brain homogenates from different sCJD subtypes (Fig. 5C).

In human prion disorders, the detection of N- and C-terminally PrP^Sc truncated fragments is a peculiarity of GSS, and, therefore, we compared the electrophoretic migration of PrP fragments detected in “anchorless PrP” Tg mice with brain samples from PrP-CAA Y145Stop and GSS F198S mutations. Under inactivating conditions, immunoblots with 12B2 of PK-digested BHs from Y145Stop, showed the presence of major bands, accounting for the monomeric 8-kDa fragment and multimeric forms thereof, whereas in F198S an additional band of 11 kDa was observed (Fig. 6A, panel a). Intriguingly, the 8-kDa band co-migrated with the faster anchorless PrP internal fragment detected in “anchorless PrP” Tg mice as well as with the synthetic peptide spanning PrP sequence 82–146. As expected, 4G11 failed to recognize internal PrP fragments (Fig. 6A, panel b). Taken together, generation of the internal PrP fragment is a feature of anchorless prions, but not of anchored prions.

Additional evidence that the 8-kDa PrP fragment detected in “anchorless PrP” Tg mice shared its electrophoretic properties with the 82–146 synthetic peptide and with the internal fragment generated by PK digestion of Y145stop and F189S mutations, was provided by results obtained following 2D-PAGE separation (Fig. 6B, panels a–f). However, whereas the 8-kDa fragment migrated as a single spot in “anchorless PrP” Tg mice, in Y145Stop and F198S mutations the internal PK-resistant fragments separated as multiple spots, within an 8.4–9.6 pI zone, as an effect of ragged N and C termini.