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. 2001 Jun;158(6):2201–2207. doi: 10.1016/S0002-9440(10)64692-5

Prion Proteins with Different Conformations Accumulate in Gerstmann-Sträussler-Scheinker Disease Caused by A117V and F198S Mutations

Pedro Piccardo *†, Juris J Liepnieks *†, Albert William *†, Stephen R Dlouhy *†‡, Martin R Farlow *§, Katherine Young *†, David Nochlin , Thomas D Bird ||, Randal R Nixon **, Melvyn J Ball **, Charles DeCarli ††, Orso Bugiani ‡‡, Fabrizio Tagliavini ‡‡, Merrill D Benson *†, Bernardino Ghetti *†‡§
PMCID: PMC1891977  PMID: 11395398

Abstract

Gerstmann-Sträussler-Scheinker disease (GSS) is characterized by the accumulation of proteinase K (PK)-resistant prion protein fragments (PrPsc) of ∼7 to 15 kd in the brain. Purified GSS amyloid is composed primarily of ∼7-kd PrP peptides, whose N terminus corresponds to residues W81 and G88 to G90 in patients with the A117V mutation and to residue W81 in patients with the F198S mutation. The aim of this study was to characterize PrP in brain extracts, microsomal preparations, and purified fractions from A117V patients and to determine the N terminus of PrPsc species in both GSS A117V and F198S. In all GSS A117V patients, the ∼7-kd PrPsc fragment isolated from nondigested and PK-digested samples had the major N terminus at residue G88 and G90, respectively. Conversely, in all patients with GSS F198S, an ∼8-kd PrPsc fragment was isolated having the major N terminus start at residue G74. It is possible that a further degradation of this fragment generates the amyloid subunit starting at W81. The finding that patients with GSS A117V and F198S accumulate PrPsc fragments of different size and N-terminal sequence, suggests that these mutations generate two distinct PrP conformers.

Prion diseases are neurodegenerative disorders present in both humans and animals. 1,2 In humans, idiopathic, genetically determined, and transmissible prion disorders have been described. 1-3 A hypothesized central event in the pathogenesis of these disorders is a conformational change of the normal membrane-associated prion protein (PrPc) into a pathogenic isoform (PrPsc). 1 PrPc is detergent soluble, protease sensitive, and has a predominantly α-helical structure. In contrast, PrPsc is insoluble in nondenaturing detergents, is relatively resistant to cleavage by proteinase K (PK), and has an increased β-sheet structure. 4-8

The methionine (M)/valine (V) polymorphism at the prion protein gene (PRNP) codon 129 can influence the clinical phenotype produced by a mutation, this is seen most markedly with the D178N mutation, which can cause Creutzfeldt-Jakob disease (CJD) or fatal familial insomnia depending on the presence of M or V at residue 129. 2 In CJD, a sporadic or inherited disease characterized by rapid progressive dementia and spongiform degeneration in the cerebrum and cerebellum, two main PrP patterns, PrPsc type 1 and type 2, have been described. 9,10 PrPsc type 1 after deglycosylation is characterized by a PK-resistant C-terminal fragment of 21 kd, whereas PrPsc type 2 is characterized by a C-terminal protease-resistant peptide of 19 kd. 9 Gerstmann-Sträussler-Scheinker disease (GSS) is a genetically determined autosomal dominant prion disease with a protracted clinical course, clinically characterized by ataxia and cognitive impairment. 3 GSS is caused by mutations P102L, P105L, A117V, G131V, F198S, D202N, Q212P, and Q217R in PRNP. 3,11,12 The pathological hallmark of GSS is the accumulation of PrP, with and without amyloid tinctorial properties, in the brain. 3 Our studies have shown that the pattern of PrPsc isoforms in the multiple GSS variants analyzed is different from that seen in CJD. 11-13 Patients with CJD present full-length and N-truncated PrP fragments whereas patients with GSS present full-length as well as N- and C-terminal truncated PrP peptides. 10-13,14,15 Previous studies showed that the amyloid subunit in GSS F198S is a 7-kd peptide with an N terminus at residue G81. 16 Similar studies in patients from one American and one Alsatian family with GSS A117V revealed that the 7-kd amyloid protein had a major N-terminal cleavage site at residue G81 and G88 to G90. 17-18 In addition, we have reported on the presence of PrPsc isoforms of ∼27 to 29, 18 to 19, and 8 kd in brain extracts and microsomal fractions of patients from the Indiana kindred with GSS F198S. 13

In GSS A117V, previous studies have been contradictory in terms of whether or not PK-resistant PrP is present. 11,19,20 The aims of the present study are to determine the biochemical characteristics of PrP in several patients and an asymptomatic carrier from two unrelated American families with PRNP A117V. 21,22 We analyzed brain homogenates, microsomal preparations, and purified PrP fractions. In addition, based on the observation that the pattern of digestion of PrPsc depends on the tertiary structure of the protein, we investigated whether PrP conformational isomers are present in phenotypically different GSS variants. To explore this possibility, we determined the N-terminal cleavage sites of the PrP fragments that accumulate in GSS A117V and GSS F198S.

Materials and Methods

The experiments were performed using brain tissue from individuals carrying PRNP mutations A117V and F198S. These individuals (seven patients and one asymptomatic carrier) derive from three families and have been studied genetically and neuropathologically. 21-26 The data on these cases is summarized in Table 1 ▶ .

Table 1.

The Clinical, Genetic, and Pathological Information for Five GSS A117V Patients and One Asymptomatic A117V Mutation Carrier; and for Two GSS F198S Patients is Summarized

Patient K AD Duration, years Residue 129 G PrP Spong NFT
Neocortex Striatum Cerebellum
1 SG 61 3 MV M +/++ ++ − − −
2 SG 39 3 MV F +/++ ++ +/− − −
3 SG 32 7 VV M +/++ +/++ +/− − −
4 SG 33 5 VV M +/++ ++ +++ − −
5 AS 45 4 VV F ++/+++ ++ +/− −/++ −
6 AS 50 N/Aa MV M − − +/− − −
7 IK 64 6 MV F +++ +++ +++ − +++
8 IK 61 12 VV F +++ +++ +++ − +++
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a, Asymptomatic carrier; K, kindred; AD, age at death; G, gender; Duration, years; duration of clinical signs in years; Spong, spongiform degeneration; NFT, neurofibrillary tangles.

Semi-quantitative analysis of lesions: −, absence; +, mild; ++, moderate; +++, severe. Subjects 1 to 6 correspond to two families with GSS A117V and patients 7 and 8 to the Indiana kindred with GSS F198S.

Biochemical Assays

Tissue was obtained from six subjects with PRNP A117V. Seven samples from the neocortex and four samples from the cerebellum of each of six individuals were analyzed. Tissue obtained from the frontal cortex of all cases was used for subcellular fractionation, including PrPsc purification and sequencing using Edman chemistry.

Tissue was obtained from the frontal cortex and cerebellum of a patient of the Indiana kindred (GSS F198S) heterozygous for the M/V polymorphism at codon 129. In addition, tissues from the frontal cortex, caudate nucleus, and cerebellum of a homozygous V 129 patient from the same kindred were also used for PrPsc purification and amino acid sequencing.

Brain tissues from a patient with sporadic CJD and from one individual dying without neurological disease were used as control.

Brain Extracts

Tissue was homogenized in 9 volumes (10% w/v) of lysis buffer (100 mmol/L NaCl, 10 mmol/L ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, 0.5% Na deoxycholate in 10 mmol/L Tris-HCl, pH 7.4). Samples were resolved in 4 to 12% acrylamide gels and transferred to nitrocellulose membranes as described. 11,13 Membranes were probed with monoclonal antibody (mAb) 3F4. 27 Immunoblot analysis for PrPsc was done after digestion of the homogenates with PK (10 μg/ml) for 1 hour at 37°C. Selected samples from patient 3 were also digested with 5, 10, 50, and 100 μg/ml of PK for 1 hour at 37°C.

Subcellular Fractionation

Membrane fractions were obtained as described. 13,15 In brief, 10% (w/v) homogenates were prepared in 0.32 mol/L sucrose, 5 mmol/L ethylenediaminetetraacetic acid, 3 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L Tris, pH 7.5. After centrifugation at 6000 × g for 10 minutes, the pellet (P1) was discarded; the supernatant (S1) was centrifuged at 100,000 × g for 1 hour at 4°C, to obtain a cytosolic fraction (S2) and a membrane fraction (P2). To determine the presence of rough endoplasmic reticulum membranes in these preparations, blots from P2 fractions were probed with mAb 9G10 (Stressgen Biotechnology, Canada) to detect GRP94, a protein normally present in the lumen of the endoplasmic reticulum membrane fraction. 28

To examine insoluble and PK-resistant PrP in the membrane fraction, the pellet (P2) was resuspended in distilled H2O, incubated on ice for 20 minutes, and centrifuged at 100,000 × g for 1 hour at 4°C. The pellet (P3) was resuspended in 150 mmol/L NaCl, 2% N-lauroylsarcosine (Sarkosyl), 25 mmol/L Tris, pH 7.4, and an aliquot removed for immunoblot analysis. The remaining sample was digested with PK at 100 μg/ml for 1 hour at 37°C. Phenylmethylsulfonyl fluoride was then added to stop PK digestion and the sample was centrifuged at 200,000 × g for 1 hour at 4°C, to obtain insoluble and PK-resistant PrP (P4). The supernatant (S4) was removed from the pellet (P4) and protein present in S4 was precipitated with methanol. The P4 and S4 fractions were resuspended in sample buffer and used for Western blot analysis.

Purification of PrPsc

PrPsc was purified as described. 29 Tissue (1.5 g) was homogenized (1:4) in 0.01 mol/L Na phosphate, pH 7.4, 10% Sarkosyl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L N-ethylmaleimide, incubated 30 minutes at room temperature and spun at 22,000 × g for 30 minutes at 10°C. The supernatant (S1) was collected and centrifuged at 215,000 × g for 2.5 hours at 10°C. The pellet (P2) was resuspended in 200 μl of 50 mmol/L Tris-HCl, pH 8.5, and 1 μl of ribonuclease A (80 μg/μl) was added. After stirring for 1 hour at 37°C, 400 μl of solution B (0.6 mol/L K iodide, 6 mmol/L Na thiosulfate, 1% Sarkosyl, 10 mmol/L Na Phosphate, pH 8.5) were added. The sample was centrifuged at 215,000 × g for 1.5 hours at 10°C through a cushion of 20% (w/v) sucrose in solution B (sucrose/sample ratio; 1:4 w/v). The pellet (P3) was resuspended in 110 μl of phosphate-buffered saline. Selected samples were used for immunoblot analysis before PK digestion. Samples from all cases were digested with PK (100 μg/ml for 1 hour at 37°C).

For deglycosylation, PrPsc was digested with 5 mU PNGase F (Glyko, Novato, CA) for 2 hours as specified by the manufacturer and analyzed by immunoblot probed with mAb 3F4. In selected samples, polyclonal antibodies AS-6800 raised against synthetic peptides corresponding to residues 89 to 104 (provided by Dr. H. Diringer) and anti PrP-95 to 108 were also used. 30 As negative control, filters were probed with the secondary antibody in the absence of primary antibody. An identical protocol was used for the analysis of PrPsc isolated from a case of CJD.

Protein Sequence Analysis

Purified PrP (ie, non-PK-treated and PK-digested samples) was resolved in 4 to 12% acrylamide gels, transferred to polyvinylidene difluoride Problott membrane (Applied Biosystems, Foster City, CA) and stained with Coomassie blue. The band of interest, as determined by comparison with a Western blot of the same material, was excised and analyzed on an Applied Biosystems model 473A protein sequencer using cycle programs provided by the manufacturer as previously described. 31

Results

PrP in Brain Extracts of Subjects Carrying The PRNP A117V

Immunoblots of brain extracts from subjects carrying the PRNP A117V mutation consistently demonstrated PrP in non-PK-treated homogenates, however considerable heterogeneity of PrPsc was observed. PrPsc varied in overall amount and in the relative quantity of the different isoforms in different brain areas of individual patients and among patients. In many samples obtained from the frontal cortex and cerebellum no PrPsc was detected (data not shown). Nevertheless, PrPsc was seen in selected samples of the frontal cortex of all patients after digestion with PK (10 μg/ml) (Figure 1 ▶ ; lanes B, D, F, H, and J), but not in samples from the asymptomatic carrier (Figure 1 ▶ , lane L). In cases 1 to 5 (Figure 1 ▶ ; lanes B, D, F, H, and J), a PrPsc band of ∼14 kd was observed, with patients 1, 2, 3, and 5 usually showing an additional band of ∼7 kd (Figure 1 ▶ ; B, D, F, and J). In some blots, a 7-kd band was also seen in patient 4. Extracts obtained from patient 3 showed 7- and 14-kd bands in samples digested with PK at concentrations ranging from 5 to 100 μg/ml (Figure 2) ▶ . For comparison, we studied in-parallel samples from CJD.

Figure 1.

Figure 1.

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Immunoblot of PrP from five patients with GSS A117V (lanes A–J), from an asymptomatic carrier (lanes K and L), and a CJD control (lanes M and N). Results from frontal cortex are shown for all cases. Samples from lanes A, C, E, G, I, K, and M were not PK treated, whereas samples from lanes B, D, F, H, J, L, and N, were PK treated. PrP was detected with mAb 3F4 and visualized by ECL. The time of exposure to film to detect the ECL product was varied to enable qualitative comparison of samples. Upper panel shows similar total PrP signals for all patients with PRNP A117V. In the lower panel lanes M, N were not included because of overexposure of the film.

Figure 2.

Figure 2.

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Immunoblot of PrP from patient 3 after digestion of frontal cortex brain homogenates with 0, 5, 10, 50, and 100 μg/ml of PK (lanes A–E, respectively). Similarly treated samples from a patient with CJD are shown in lanes F–J. Different times of exposure (1 second, 10 seconds, and 30 seconds) were obtained. PrP was detected with mAb 3F4 and visualized by ECL.

PrP in Subcellular Fractions of Patients with PRNP A117V

PrP partitioned with membranes in samples obtained from the frontal cortex of subjects carrying the PRNP A117V mutation. Examples from two clinically affected and one asymptomatic carrier are shown in Figure 3 ▶ ; lanes A, D, and G. We observed that the membrane fraction obtained from the affected subjects (1 to 5) contained detergent-insoluble and PK-resistant PrP (ie, PrPsc) fragments of 7 and 14 kd. PrPsc present in patients 3 and 4 is shown in Figure 3 ▶ , lanes B and E. Figure 3 ▶ also shows that PrPsc was not detected in the soluble fraction of these patients. Soluble fractions obtained from patients 3 and 4 are shown in Figure 3 ▶ , lanes C and F. PrPsc was not detected in membranes or in the soluble fractions of the sample obtained from the frontal cortex of the asymptomatic A117V mutation carrier (Figure 3 ▶ , lanes H and I). To determine the presence of microsomes, immunoblots were probed with mAb 9G10, directed against the heat-shock protein GRP 94 normally present in the lumen of the endoplasmic reticulum membranes. A positive result was obtained (not shown).

Figure 3.

Figure 3.

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Immunoblot of PrP in membrane fractions obtained from the frontal cortex of patient 3 (lane A), patient 4 (lane D), and from an asymptomatic carrier (lane G). Detergent-insoluble and PK-resistant PrP of ∼7 and 14 kd are observed in patients 3 and 4 (lanes B and E), but not in the asymptomatic carrier (lane H). No PrPsc is observed in the PK-digested soluble fraction in patients or carrier (lanes C, F, and I). PrP was detected with mAb 3F4 and visualized by ECL.

Partially Purified PrP in Subjects Carrying the PRNP A117V Mutation

Because of the variability of detecting abnormal PrP in total brain homogenates and to the fact that previously published protocols are effective in concentrating relatively pure PrPsc, we analyzed partially purified fractions obtained from the frontal cortex of patients 1 to 5 and from the asymptomatic carrier (case 6). After PK digestion, two major bands of 14 and 7 kd were detected in the patients (ie, patients 1 to 5). A similar pattern was seen when immunoblots were probed with a panel of antibodies raised against the mid-region of PrP (ie, PrP residues 89 to 112), however no signal was seen when the primary antibody was omitted. An example of PrPsc banding pattern in patient 5 is shown in Figure 4 ▶ , lane B. No PrPsc was observed in the partially purified sample obtained from the asymptomatic carrier (not shown).

Figure 4.

Figure 4.

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Immunoblot of purified PrP obtained from the frontal cortex of patient 5 (lanes A–C) and from a CJD control (lane D). Samples were either nontreated, ie, no PK or PNGase F treatment (lane A); treated with PK (lanes B and D); or PK- and PNGase F-treated (lane C). PrP was detected with mAb 3F4 and visualized by ECL.

Analysis of Glycosylation State of PrPsc Fragments

In CJD and some other prion diseases, PrPsc is composed of a C-terminal-glycosylated PK-resistant core. The 7-kd PrPsc fragment seen in GSS A117V is detected with mAb 3 F4. Considering the apparent size and immunological profile of this peptide (ie, containing residues 109 to 112, as shown by mAb 3F4 immunoreactivity), we speculated that this peptide would not contain the C-terminal N-linked glycosylation sites at residues 181 and 197. To explore this possibility further, we performed enzymatic deglycosylation on samples obtained from all patients (ie, patients 1 to 5), and showed that an identical mobility is seen in PrPsc peptides before and after PNGase F treatment. Figure 4 ▶ , lane C, shows a representative sample obtained from patient 5. This finding supports the concept that glycosylation sites are not present.

PrP Fragments in Non-PK-Treated Samples

We noticed that in selected samples the low-molecular weight PrP fragments (ie, 7 and 14 kd) could be seen before PK digestion. To analyze if the truncated fragments were insoluble in nondenaturing detergent we partially purified PrP in non-PK-digested samples (through ultracentrifugation in the presence of Sarkosyl). In all patients (ie, patients 1 to 5), we observed bands of ∼27 to 35, 14, and 7 kd. A sample from patient 5 is shown in Figure 4 ▶ , lane A. The stoichiometry of the low-molecular weight fragments is different between PK- and non-PK-digested samples. PK digestion increases the amount of the 7- and 14-kd bands. The similar size of the 7-kd fragments in non-PK-treated and PK-treated samples suggest that a similar or identical cleavage site may be present in vivo and in vitro.

Determination of the N-Terminal Cleavage Site of PrP Fragments in Patients with GSS A117V and GSS F198S

To characterize the primary structure of the 7-kd PrPsc fragment, partially purified and PK-treated PrP obtained from the frontal cortex of patients with GSS A117V (ie, patients 1 to 5) was analyzed using Edman chemistry, after resolution of fragments by gel electrophoresis (see Materials and Methods). Sequence analysis of the 7-kd peptide from patient 3 (for 20 cycles) yielded GQGGGTHSQWNKPS KPKTNM as the major N-terminal sequence corresponding to residues 90 to 109 of PrP. The data were consistent with minor amounts of PrP fragments starting with residues 86, 88, and 92. Analysis of the 7-kd band from the other four patients showed that the major sequence also started with residue 90 of PrP. As stated above, a 7-kd band was also seen in immunoblots obtained from non-PK-digested samples. Sequence analysis of this 7-kd band from patient 4 yielded GXGQGGGTHSQXNKP as the N-terminal sequence corresponding to residues 88 to 102 of PrP.

Patients with GSS F198S and GSS A117V accumulate PrPsc fragments that can be cleaved by PK to generate peptides of different mobility (ie, the small fragment is 7 kd in GSS A117V and 8 kd in GSS F198S) in gel electrophoresis. This observation could be because of the presence of conformational isomers that expose different PrP residues to hydrolysis by PK in the presence of detergents. To explore this possibility, we analyzed the 8-kd PrPsc fragment obtained from the frontal cortex of two patients with GSS F198S (Figure 5) ▶ . Sequence analysis for 29 cycles yielded GQPHGGGWGQPHGGGWGQGGGTHSQWNKP corresponding to residues 74 to 102 of PrP in both cases. This indicates PK cleavage in the second PrP octarepeat region for GSS F198S, different from the PK cleavage site for GSS A117V.

Figure 5.

Figure 5.

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Immunoblot of purified PrP obtained from the frontal cortex and cerebellum of patient 8 (lanes B and C), from the cerebellum of patient 7 (lane D), and from a CJD control (A). Identical banding pattern was observed in samples obtained from patients 7 and 8. Samples were treated with PK (lanes A–D). PrP was detected with mAb 3F4 and visualized by ECL.

To analyze the possibility that different PrP conformers might be present in different brain areas, we determined the N-terminal sequence of PrPsc purified from the caudate nucleus (an area with absent or small amounts of amyloid), and the cerebellum (a region with abundant amyloid) in a homozygous 129 V patient with GSS F198S. Similar results through 10 to 15 cycles were observed in both samples indicating G58, G66, or G74 as the N terminus. Although we were not able to determine sufficient residues on the fragments to identify the exact N terminus, based on the size and immunological profile, the simplest interpretation of the data are that the major N terminus is at residue 74, as found in the frontal cortex.

Discussion

This study demonstrates that PrPsc is present in all five patients with GSS A117V but was not detected in the samples analyzed from the asymptomatic carrier. The 14- and 7-kd PrP species were observed in brain extracts and microsome preparations. Whether PrPsc partitioning with membranes is due to protein-protein interaction, protein-lipid interaction, or the formation of sedimentable aggregates in the presence of detergents remains undetermined. PK-resistant fragments of 14 and 7 kd were also seen in purified PrPsc fractions. In all patients (1 to 5), the sequencing of the 7-kd peptide in non-PK- and PK-digested samples, showed a major N-terminal cleavage site between G87 to G88 and W89 to G90, respectively.

It is noteworthy that PK digestion changed the stoichiometry of the various PrP peptides observed in the undigested preparations with an increase in the 14- and 7-kd fragments and disappearance of the high-molecular weight isoforms of ∼27 to 35 kd. These findings indicate that these patients (1 to 5) accumulate larger PrP isoforms that can be cleaved to smaller, insoluble, PK-resistant fragments. The detection of low-molecular weight peptides in samples that have not been treated with PK, suggest that truncated PrP is generated in vivo by hydrolysis of pre-existing intermediates. These peptides may be the result of the metabolism of PrP in a pathway associated with amyloid formation, because similarly sized fragments have been found in isolated amyloid cores of patients with GSS. 16-18

These data differ from those showing the absence of PrPsc in patients with GSS A117V. 20 It has been proposed that a transmembrane PrP isoform, detected after limited PK digestion at low temperature in the absence of ionic detergents, plays a central role in the pathogenesis of GSS A117V. 20 We did not investigate the presence of different topological forms of PrP. Thus, we cannot determine whether the PrPsc present in the patients analyzed by us (1 to 5) corresponds to the transmembrane, extracellular, secreted, or membrane bound via a phosphatidylinositol anchor PrP species. It is important to note that the amount of PrPsc in the patients (1 to 5) reported here is significantly smaller than that found in other GSS variants. 11,13 In agreement with the results presented here, recent studies in an Alsatian patient with GSS A117V 18 showed that the major component of amyloid fibrils was a ∼7-kd peptide with ragged N terminus starting mainly at G88 and G90. 18

The 14- and 7-kd bands, seen in GSS A117V (1 to 5), are detected by antibodies directed to the mid-region of PrP indicating that these fragments include the intact epitope recognized by mAb 3F4 consisting of residues 109 to 112. It has been shown that in a normal human brain, PrPc is endogeneously cleaved at residues H111 or M112. 15 Thus, our results suggest that patients with GSS A117V have an alternative metabolic pathway, leading to the accumulation of N- and C-truncated PrP fragments with abnormal physicochemical properties. The data also suggests that the proteolytic pathway present in these patients with GSS is different from that previously described in CJD. 10,15 In the latter, N-truncated, C-terminally-intact PrPsc fragments accumulate in the brain. 10,15

To determine whether the ∼14-kd fragment could correspond to a glycoform of the ∼7-kd peptide, we performed enzymatic deglycosylation and observed no shift in electrophoretic mobility, suggesting that the larger fragment is either nonglycosylated or that sugars at residues N181 and N197 are not accessible to enzymatic cleavage. Whether the ∼14-kd band is an oligomer of the 7-kd peptide or a larger fragment with different N- and C-termini remains to be investigated.

Studies on amyloid fractions have previously shown that patients with GSS F198S accumulate amyloid peptides of ∼11 kd spanning residues 58 to 150. 32 Further analysis showed that the smallest amyloid subunit in GSS F198S and GSS Q217R corresponds to a 7-kd fragment comprising residues W81 to Y150 and W81 to E146, respectively. 16 In addition, preliminary data on a patient of an American family with GSS A117V showed a similar fibrillogenic fragment with a ragged N terminus corresponding to W81, G82, and Q83 and the C terminus at E146. 17 Thus, patients with these GSS variants may accumulate amyloid subunits of similar size and primary structure, despite the different genotypes and phenotypic presentations.

We hypothesized that the purification of PrPsc may allow the isolation of 1) PrP peptides that have not acquired fibrillogenic properties, 2) peptides that are not metabolized in an amyloidogenic pathway, or 3) peptides that are precluded from the extraction procedure used to isolate amyloid cores. Therefore, to expand our studies we purified PrPsc from patients of the Indiana kindred with GSS F198S and determined the N-terminal cleavage site of the ∼8-kd fragment isolated from areas with and without amyloid accumulation. In all of the samples analyzed the major N-terminal cleavage site corresponded to residue G74 of the octapeptide repeat region, suggesting that as yet unidentified local factors may contribute to PrP amyloidogenesis. In view of the fact that the smallest amyloidogenic fragment has a mobility of ∼7 kd and N terminus at W81 and PrPsc peptides of ∼8 kd have an N terminus at G74, we speculate that sequential proteolytic cleavage of a precursor PrPsc fragment generates fibrillogenic peptides in GSS F198S.

In conclusion, the data obtained in the GSS variants analyzed in this study demonstrate that N- and C-truncated PrP isoforms of different size and N-termini, accumulate in GSS A117V and GSS F198S. Based on the principle that the pattern of digestion (ie, protease digestion in the presence of detergents) depends on the tertiary structure of proteins, 33 the data suggest that PrP conformational isomers are present in these patients.

The results show that octarepeats 3 and 4 are an integral part of the 8-kd peptide present in GSS F198S, but not in the 7-kd fragments detected in patients with GSS A117V. The importance of the accumulation of peptides with different N-termini in these GSS variants is unclear at this time. However, other investigators have suggested that Cu2+ binds to a structure defined by two of the octarepeats in PrP containing the sequence PHGGGWGQ. 34 They proposed that this binding could induce conformational changes in PrP. 34 In addition, short peptides corresponding to the octapeptide repeat motif of PrP have been reported to bind Cu2+. 35,36 Moreover, it has been shown that PrP fragments can be transformed from a predominantly α-helical monomeric form to an oligomeric β-sheet-rich secondary structure. 37,38 Therefore, PrPsc fragments with a distinct structure could have different neurotoxic properties or a tendency to form aggregates, providing a possible mechanism underlying the differences in phenotypic presentation among GSS variants.

Acknowledgments

We thank Bradley S. Glazier for editorial assistance.

Footnotes

Address reprint requests to Pedro Piccardo M.D., Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Dr., MS A142, Indianapolis, IN 46202-5120. E-mail: ppiccard@iupui.edu.

Supported in part by Public Health Service R01 NS 29822 and P30 AG10133; and the Italian Ministry of Health, Department of Social Services (RF 97-13 and 99-38).

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