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J. Biol. Chem., Vol. 279, Issue 37, 38936-38942, September 10, 2004
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From the
Departments of Neurological and Visual Sciences, Section of Neurology and ¶Agricultural and Industrial Biotechnologies, University of Verona, 37134 Verona, Italy and the Department of Pathology, New York University Medical Center, New York, New York
Received for publication, May 17, 2004 , and in revised form, July 7, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 kDa (6, 7). Conversely, PrPSc is cleaved within the N-flexible region with formation of a fragment referred to as C2, sharing its conformation with PrP27-30 (6, 7). Recently, C-terminal PrP fragments of 11-12 kDa and 12-13 kDa have been identified in iatrogenic (8) and sporadic Creutzfeldt-Jakob disease (sCJD) (8, 9).
Neuropathological hallmarks of transmissible spongiform encephalopathies include spongiosis, neuronal loss, and gliosis accompanied by PrPSc deposition at synaptic locations and at the rims of vacuoles or as large extracellular amyloid plaques and plaque-like aggregates (10). Different patterns of PrP deposition and intracerebral regional distribution of lesions are explained by the existence of prion strains with identifiable biological properties and distinctive physicochemical features of PrPSc (1).
sCJD has an annual incidence of 1-1.5 per million worldwide and a still unknown etiology; prevailing hypotheses suggest that the disorder is triggered by pathogenic somatic mutations in the prion protein gene (PRNP) or by random spontaneous changes in PrPC conformation (11). However, there is concern that some sCJD cases could be secondary to environmental exposure, case-to-case transmission, or unnoticed food chain contamination, as suggested by the occurrence of atypical phenotypes, spatiotemporal disease clusters (12), and the increased incidence of the disorder recently observed in Switzerland (13). Because of the variability in clinical presentation, signs at evolution, and neuropathological changes, the disorder with the double eponym Creutzfeldt-Jakob should be properly called syndrome rather than disease. Different sCJD phenotypes largely correlate with the PRNP codon 129 genotype, a site of methionine or valine polymorphism, and distinct PrPSc types (14) currently classified according to the molecular mass of their PrP27-30 core fragment in turn reflect the tertiary structure of PrPSc. However, controversial results have been obtained by different authors who have proposed the classification of sCJD PrP27-30 into two (14) and four types (15). Two main limitations of these studies are (i) the use of a relatively crude technique of protein separation, such as SDS-PAGE, and (ii) the detection of PrP species with an antibody recognizing the N-terminal 109-112 epitope, which precludes the recognition of PrP molecules lacking this region.
Here we studied 32 subjects, with definite sCJD of all genotypes at PRNP codon 129 and with different PrPSc types, to characterize C-terminal PrPSc fragments in distinct disease phenotypes and host genotypes. Brain homogenates resolved by SDS-PAGE and two-dimensional electrophoresis were immunoblotted with anti-PrP antibodies recognizing epitopes located in the N-terminal and C-terminal regions. Here we report the detection of novel truncated PrPSc species in association with distinct PrP27-30 types. In addition, we provide evidence that sCJD phenotypes with PrP amyloid plaques or plaque-like deposits are associated with pathological PrP species matching the so-called C1 PrPC fragment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mercaptoethanol, 2 mM EDTA, 10% glycerol, 62.5 mM Tris, pH 6.8) and boiled for 5 min. An equivalent of 0.4 mg of wet tissue was loaded onto 12% SDS-PAGE gels and then transferred onto polyvinylidene difluoride membrane (Immobilon P, Millipore) for 2 h at 60 V. The 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 appropriate antibodies. Blots were developed with an enhanced chemiluminescence system (ECL, Amersham Biosciences), and PrP was visualized on autoradiography film (Hyperfilm, Amersham Biosciences). The following anti-PrP monoclonal antibodies were used: 3F4, recognizing human PrP residues 109-112 (1:30000); 6H4, binding an epitope between amino acids 144-152 (Prionics, 1:5000), and SP-214, directed against the 214-231 sequence of human PrP (1:1000) (7).
Two-dimensional Gel Electrophoresis—For isoelectric focusing, using immobilized pH gradients in the first dimension, 8-cm long precast gels with a linear pH range of 3-10 were used. Before isoelectric focusing, the dry gels were swollen for 16 h in 125 µl of buffer (8 M urea, 5% -mercaptoethanol, 2% Nonidet P-40, and 2% ampholyte) containing 10 µl of sample (an equivalent of 1 mg of wet tissue). Isoelectric focusing was carried out at 20 °C for 30 min with 500 V, 30 min with 1000 V, and 1 h with 4000 V in a cooled horizontal electrophoresis unit (IPGphor, Amersham Biosciences). For the second dimension, the immobilized pH gradient strips were equilibrated for 15 min in 50 mmol/liter Tris-HCl, 6 mol/liter urea, 10% glycerol, 2% SDS, and a trace of bromphenol blue and loaded in a 12% SDS-PAGE as described previously (17). Immunoblotting was performed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In marked contrast, following PK digestion and immunoblot with 6H4, sCJD brains from subjects MV and VV at codon 129 and with type 2 PrPSc contained only the PrPSc triplet (Fig. 1B, lane 2) but not the 16-17-kDa fragment found in cases with type 1 PrPSc and in MM2 cases. However, after PNGase treatment, PK-digested samples from MV2 and VV2 cases showed an additional band migrating at 18 kDa (Fig. 1B, lane 4).
Finally, immunoblot with SP-214 of brain homogenates from different sCJD subtypes showed, in addition to the PrPSc triplet, several PK-resistant PrPSc bands, with the lowest molecular mass fragments being detected at 12 kDa. The pattern of PK-resistant PrP species obtained in cases with type 1 PrPSc disclosed indistinct banding in a range between 12-17 kDa, either prior to (Fig. 1C, lane 1) or after deglycosylation (Fig. 1C, lane 3), whereas in MV2 and VV2 subjects (Fig. 1C, lane 2), fewer truncated fragments were seen, migrating at 18- and 12-kDa zones after PNGase and PK treatment (Fig. 1C, lane 4).
Effect of pH on PrP27-30 and on Truncated PrP Fragments—In a previous study, we observed that one type of PrPSc, detected in the classical form of sCJD, changes its conformation after exposure to acidic and basic pH values (18). This property, likely dependent on the presence of octapeptide repeats in the PrPSc molecule, enabled us to distinguish two PrPSc types that share an identical electrophoretic mobility after solubilization in lysis buffer at pH 7.4. Following immunoblot with 3F4, the PK-resistant fragment of 21 kDa, detected in 9 MM subjects and in 1 VV case, migrated slower at pH 5 as compared with the migration observed when samples were solubilized at pH 8 (Table I). In contrast, PrPSc migration was not consistently modified in 6 MM, 1 MV, and 1 VV cases with type 1 PrP27-30 and in all cases with type 2 PrP27-30. To further clarify the influence of the octapeptide region in determining sensitivity to pH, brain homogenates from each single case of the group sensitive to pH were exposed to consecutive PK digestions, first to acidic and subsequently to basic pH. As expected, the immunoblots with 6H4 showed that, at acidic pH, the main fragment migrated at 22.5 kDa, and following further digestion at pH 8, it was detected in a 20-kDa zone because of the effect of an additional cleavage. Immunoblots with SP-214, recognizing the 214-231 C-terminal sequence of human PrP, showed similar results (data not shown) thus suggesting that the 22.5 and 20 kDa bands differ at the N terminus and not at the C terminus. Conversely, the electrophoretic migration of the C-terminal fragment of 16-17 kDa was unaffected by serial acidic and basic digestion procedures (Fig. 1D, lanes 1 and 2), confirming the role of the octapeptide region in conferring PrPSc susceptibility to conformational changes.
Two-dimensional mapping of PrP isoforms in control and sCJD brains—Recently, we provided a detailed two-dimensional mapping of PrPC glycoforms by using the 3F4 antibody, recognizing a linear epitope expressed in the full-length PrPC and in the C2 fragment (17). When analyzing PrPC in two dimensions, the customary di-, mono-, and unglycosylated bands of the protein, obtained after SDS-PAGE, are separated in several spots according to two independent features: the isoelectric point (which reflects the net charge) and the mobility (determined by the molecular mass). In addition, after two-dimensional separation, each spot can be assigned to glycosylated or unglycosylated species based on its shape, configuration, tail, and smear. Although the 6H4 antibody was unable to detect PrPC molecules after two-dimensional separation of control brain homogenates, the SP-214 monoclonal antibody revealed several glycosylated and unglycosylated PrP forms with faster migrating species being detected at 18 kDa, corresponding to unglycosylated C1 isoforms (Fig. 2A). Negative results obtained with 6H4 suggest that this antibody recognizes a conformational rather than a linear PrP epitope and that this epitope is lost after solubilization of PrP in high molar urea. After PNGase treatment, truncated PrPC glycoforms were all reduced into several spots migrating at 20- and 18-kDa zones (Fig. 2B), corresponding to PrP fragments referred to as C2 and C1, respectively. Differing from controls, each single homogenate of cortical areas, subcortical nuclei, and cerebellum from sCJD brains showed increased amounts of C2 (migrating at 21 kDa in cases with type 1 PrPSc and 19 kDa in those with type 2 PrPSc) and the presence of additional acidic PrP glycoforms and low molecular mass unglycosylated species. The composition, electrophoretic migration, and relative abundance of these PrP species were different among distinct sCJD subtypes. In particular, sCJD cases of all codon 129 genotypes with type 1 PrPSc were characterized by the increased expression of unglycosylated PrP forms in a 12-17-kDa zone as opposed to the low abundance of these species in the MM2 subjects and the prominence of low molecular mass acidic PrP glycoforms in MV2 and VV2 subtypes (Fig. 2C). Interestingly, truncated PrP species observed in different sCJD subtypes were partially recovered in the detergent-insoluble fraction from different brain areas (Fig. 3, A-C).
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16-17- and 12-14-kDa zones, pI 4-5 (Fig. 4, B, D, and F). Some variability was observed among individual cases in the relative intensity and representation of these spots, but their migration and configuration were highly comparable in all cases. Additionally, slight quantitative but not qualitative differences were seen in different brain regions from each case. After deglycosylation and PK-treatment, major glycoforms of 21-30 kDa were reduced to 21 kDa, pI 6.6-7.2, whereas lower molecular mass species were detected at 16-17- and 12-14-kDa zones, pI 4.5-5.5 (Fig. 4, B', D', and F').
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Finally, in the MV2 and VV2 groups, major 3F4-positive glycoforms of 29, 25, and 19 kDa (Fig. 4I) were associated with small amounts of 12-14-kDa species recognized by the SP-214 antibody (Fig. 4L). However, after glycan removal, a set of spots migrating at 17.5-18 kDa was detected in cortical and subcortical brain regions (Fig. 4L'). Based on their pI values and apparent molecular masses, these spots matched the corresponding C1 PrPC peptides observed after deglycosylation of normal brain homogenates (see Fig. 2B).
These results indicate that in MV2 and VV2 cases, glycosylated C1 isoforms acquire PK-resistance, although the possibility that C1-sized fragments are generated upon protease treatment cannot be ruled out. On the contrary, unglycosylated isoforms of C1 detected in both normal and pathological brain homogenates (see Fig. 2, A and C) lack detergent insolubility (see Fig. 3C) and PK-resistance (see Fig. 4L) in sCJD brains. The schematic (Fig. 5) depicts different patterns of truncated PrP fragments found in association with distinct molecular sCJD subtypes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using sensitive protein separation techniques and Western blot with antibodies recognizing the C-terminal globular domain of prion protein, we have identified new protease-resistant PrP species in brain homogenates from sCJD subjects. These new fragments, detected in the mass interval between 16 and 18 kDa, showed two distinct and highly reproducible electrophoretic patterns, each segregating preferentially with type 1 or type 2 PrP27-30. The electrophoretic migration of these newly identified PK-resistant PrP products is, therefore, intermediate between the unglycosylated PrP27-30 fragment (which is currently used to classify molecular PrPSc types) and the 12-14 kDa PrP molecules observed in all sCJD subtypes and named CTF 12/13 (9). The immunoblot detection of low molecular mass PrP fragments in untreated whole brain homogenates from sCJD subjects indicates that such fragments are generated by an endogenous proteolytic mechanism. It should also be emphasized that the detergent insolubility of these molecules and the lack of shift in their apparent molecular weight after PK digestion support the view that these PrP species adopt a fully protease-resistant conformation in vivo.
Taken together, the present findings point out that multiple abnormal PrP conformers are expressed during the spreading of sCJD strains. This could result from diverse subcellular sites of formation or, alternatively, from the generation of PrP conformers from full-length and N-terminally truncated PrPC precursors. Furthermore, the identification of the 16-17- and 12-14-kDa forms in subjects of all genotypes with type 1 or MM with type 2 PrPSc suggests that the mechanism involved in the formation of these forms is not influenced by codon 129 and by the type of PrP27-30. Although the three classes of PrP fragments characterized in the present study show biochemical properties of pathological PrP, it remains to be determined if they maintain all of the biological properties of PrPSc.
A further implication of the identification of 16-17-kDa forms is related to the molecular classification of PrPSc types encountered in sCJD. The influence of several experimental conditions, including pH and EDTA concentration, on the conformation of type 1 PrPSc and, therefore, on its electrophoretic migration is well known (18, 19). On this basis, we propose the use of conventional Western blot with 6H4 antibody, which is able to detect an accompanying fragment of 16-17 kDa in all cases with type 1 PrPSc. Such a standardized molecular classification of sCJD would have major implications for epidemiological studies, whereas more sophisticated investigations, such as two-dimensional mapping and PrP sequencing, should be reserved for atypical cases.
To date, the existence of distinct strains of the agent responsible for human prion diseases has been difficult to explain with just the biochemical findings of two main PrP27-30 conformations and with microsequencing analyses showing two truncation patterns in sporadic, inherited, and acquired diseases (21). It is well known that glycans may play a role in the phenotypic determination of some prion conditions, and their role has been recently proposed to explain differences between sporadic fatal insomnia and MM2 sCJD, two conditions sharing PrP27-30 type and PRNP codon 129 (22). However, different glycosylation states of PrPSc cannot fully account for prion diversity. Recent transmission studies of sCJD to transgenic mice show three distinct incubation periods of different sCJD subtypes (23). In particular, inocula from subjects MM1 and MV1 have the same incubation times, and, therefore, these molecular types appear as a homogeneous group. MV2 and VV2 cases, when transmitted to mice with Val at position 129, induce the disease at the same post-inoculum intervals and thus suggesting similar biological properties of these sCJD strains. On the other hand, MM2 inocula show heterogeneous transmission properties and incubation times different from the above groups.
Based on the biochemical patterns obtained by combining the PrP27-30 core fragment and lower truncated PrP species, here we show three distinct groups of disease-associated PrP species in sCJD with type 1 PrPSc, MM2 subjects, and MV2/VV2 cases. Strikingly, similar molecular groups of PrP species are obtained by Satoh et al. (8) when combining truncated fragments of 11-12 kDa with type 1 and type 2 PrP27-30. Whether the present biochemical findings represent a molecular basis for different biological properties of the above sCJD strains remains to be determined.
Despite the recognition of conformational variants of disease-associated PrP species in experimental and naturally occurring prion diseases, little is known about the influence of different molecules on disease phenotype (24). Although it is commonly believed that PrP27-30 has a major role in modulating sCJD phenotype, it would be of interest to learn whether different truncated PrPs are related to specific clinical and pathological traits. Previous studies have disclosed that an internal PrP fragment of 7-8 kDa detected in patients with P102L Gerstmann-Sträussler-Scheinker disease (7, 25) and a C-terminal fragment associated with familial Creutzfeldt-Jakob disease due to E200K mutation (26) are toxic to neurons in combination with or in the absence of PrP expression (27, 28). Here we have found that a fully glycosylated PrP fragment, closely matching the C1 PrPC fragment, was associated with amyloid plaques in MV2 cases and plaque-like PrP deposits in VV2 subjects. On the contrary, C1 PrPSc was not observed in sCJD subtypes lacking extracellular PrP deposition. It is noteworthy to emphasize that we also determined2 the presence of C1 PrPSc in subjects with variant Creutzfeldt-Jakob disease (a condition characterized by florid PrP amyloid plaques) as well as in the newly identified bovine amyloidotic spongiform encephalopathy (BASE) (3) but not in amyloid-free typical bovine spongiform encephalopathy (BSE). Earlier studies have shown that C1 is a cell surface glycophosphoinositol-anchored protein starting at amino acid 110/111 in human neuroblastoma cells (6) and present under multiple truncation forms in brain tissue (17). According to our findings, it is conceivable that extracellular release of C1 PrPSc could be responsible for PrP deposition and aggregation, as observed in MV2 and VV2 sCJD subtypes. Altogether, these results show that PrP-truncated fragments may influence the disease phenotype of sCJD and that their characterization is important for the molecular definition of sCJD subtypes.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Neurological and Visual Sciences, Section of Neurology, Policlinico G. B. Rossi, P.le L. A. Scuro, 10, 37134 Verona, Italy. Tel.: 39-045-8074286; Fax: 39-045-585933; E-mail: salvatore.monaco{at}mail.univr.it.
1 The abbreviations used are: PrPC, cellular prion protein; PrPSc, scrapie isoform of prion protein; sCJD, sporadic Creutzfeldt-Jakob disease; MM, diglycosylated PrPSc species; MV, monoglycosylated PrPSc species; VV, unglycosylated PrPSc species; PK, proteinase K.
2 G. Zanusso, A. Farinazzo, and S. Monaco, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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