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Effects of Different Experimental Conditions on the PrPSc Core Generated by Protease Digestion

2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês

10.1074/jbc.m313220200

ISSN

1083-351X

Autores

Silvio Notari, Sabina Capellari, Armin Giese, Ingo M. Westner, Agostino Baruzzi, Bernardino Ghetti, Pierluigi Gambetti, Hans A. Kretzschmar, Piero Parchi,

Tópico(s)

Viral Infections and Immunology Research

Resumo

The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure. The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure. Transmissible spongiform encephalopathies (TSEs), 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; PrP, prion protein; PrPc, cellular prion protein; PrPSc, scrapie/protease-resistant prion protein; PK, proteinase K; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; vCJD, variant CJD; PRNP, prion protein gene; MM1, methionine homozygote at codon 129, PrPSc type 1; MV1, methionine/valine heterozygote at codon 129, PrPSc type 1; VV1, valine homozygote at codon 129, PrPSc type 1; MM2, methionine homozygote at codon 129, PrPSc type 2; VV2, valine homozygote at codon 129, PrPSc type 2; MV2, methionine/valine heterozygote at codon 129, PrPSc type 2; LB, lysis buffer; PBS, Dulbecco's phosphate-buffered saline lacking Ca2+ and Mg2+. 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; PrP, prion protein; PrPc, cellular prion protein; PrPSc, scrapie/protease-resistant prion protein; PK, proteinase K; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; vCJD, variant CJD; PRNP, prion protein gene; MM1, methionine homozygote at codon 129, PrPSc type 1; MV1, methionine/valine heterozygote at codon 129, PrPSc type 1; VV1, valine homozygote at codon 129, PrPSc type 1; MM2, methionine homozygote at codon 129, PrPSc type 2; VV2, valine homozygote at codon 129, PrPSc type 2; MV2, methionine/valine heterozygote at codon 129, PrPSc type 2; LB, lysis buffer; PBS, Dulbecco's phosphate-buffered saline lacking Ca2+ and Mg2+. or prion diseases, are a phenotypically heterogeneous group of neurodegenerative disorders that affects humans and animals. Human diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease, kuru, and fatal insomnia (1Parchi P. Clark C. Trojanowski J.Q. Neurodegenerative Dementias: Clinical Features and Pathological Mechanisms. McGraw-Hill Inc., New York2000: 341-365Google Scholar, 2Ghetti B. Bugiani O. Tagliavini F. Piccardo P. Dickson D. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. ISN Neuropath Press, 2003: 318-325Google Scholar, 3Ironside J.W. Head M.W. Will R.G. Dickson D. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. ISN Neuropath Press, 2003: 310-317Google Scholar). CJD, by far the most common human TSE, may occur as a sporadic disease of unknown etiology (sCJD), a disease associated with mutations in the prion protein gene (PRNP), or a proven exogenous infection. The latter group includes variant CJD (vCJD) a distinct disease phenotype that is believed to have been transmitted from cattle to humans through the consumption of contaminated meat (3Ironside J.W. Head M.W. Will R.G. Dickson D. Neurodegeneration: The Molecular Pathology of Dementia and Movement Disorders. ISN Neuropath Press, 2003: 310-317Google Scholar). The cellular prion protein (PrPc), a host-encoded, copper- and membrane-bound glycoprotein of unknown function, has a key role in TSE pathogenesis (4Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5101) Google Scholar). There is no agent replication or transmission of infectivity in the absence of PrPc expression (5Büeler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1802) Google Scholar). Moreover, an abnormal, partially protease-resistant isoform (PrPSc) of PrPc specifically accumulates in the nervous system during infection and represents the hallmark of the disease (4Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5101) Google Scholar, 6Bolton D.C. McKinley M.P. Prusiner S.B. Science. 1982; 218: 1309-1311Crossref PubMed Scopus (1009) Google Scholar). The conversion of PrPc to PrPSc is a posttranslational event associated with an increase in β-sheet secondary structure in the protein (7Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2064) Google Scholar, 8Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar). CJD, like scrapie of sheep, comprises a broad spectrum of clinicopathological variants (9Parchi P. Giese A. Capellari S Brown P. Schulz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar). In scrapie transmitted to mice, the most extensively studied TSE, the strain of the agent and the host genotype variability determined by polymorphisms in the coding region of PRNP are the major determinants of disease phenotype (10Bruce M.E. McConnell I. Fraser H. Dickinson A.G. J. Gen. Virol. 1991; 72: 595-603Crossref PubMed Scopus (338) Google Scholar). Different prion strains transmit diseases to syngenic animals that differ in topography, type of lesion, and intracerebral distribution of PrPSc. 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Because the analysis of biochemical properties of PrPSc is much less time-consuming than bioassays in mice, unraveling the physicochemical properties of PrPSc associated with each TSE strain or phenotype (i.e. PrPSc "typing") has undoubtedly become of crucial importance for strain typing and molecular classification of TSEs, with wide implications for both disease diagnosis and epidemiologic surveillance. Unfortunately, there is a significant disparity in the literature regarding the existence of distinct human PrPSc types (23Parchi P. Capellari S. Chen S.G. Petersen R.B. Gambetti P. Kopp N. Brown P. Kitamoto T. Tateishi J. Giese A. Kretzschmar H. Nature. 1997; 386: 232-233Crossref PubMed Scopus (231) Google Scholar, 24Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar, 25Parchi P. Zou W. Wang W. Brown P. Capellari S. Ghetti B. Kopp N. Schulz-Schaeffer W.J. Kretzschmar H.A. Head M.W. Ironside J.W. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10168-10172Crossref PubMed Scopus (266) Google Scholar, 26Wadsworth J.D.F. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar, 27Zanusso G. Farinazzo A. Fiorini M. Gelati M. Castagna A. Righetti P.G. Rizzuto N. Monaco S. J. Biol. Chem. 2001; 276: 40377-40380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and this to some extent is also true for animal TSEs (28Hope J. Wood S.C. Birkett C.R. Chong A. Bruce M.E. Cairns D. Goldmann W. Hunter N. Bostock C.J. J. Gen. Virol. 1999; 80: 1-4Crossref PubMed Scopus (144) Google Scholar, 29Baron T.G. Madec J.Y. Calavas D. Richard Y. Barillet F. Neurosci. Lett. 2000; 284: 175-178Crossref PubMed Scopus (74) Google Scholar, 30Kuczius T. Groschup M.H. Mol. Med. 1999; 5: 406-418Crossref PubMed Google Scholar). Based on differences in gel mobility and N-terminal sequence of the core fragments generated by proteinase K (PK), Parchi et al. (23Parchi P. Capellari S. Chen S.G. Petersen R.B. Gambetti P. Kopp N. Brown P. Kitamoto T. Tateishi J. Giese A. Kretzschmar H. Nature. 1997; 386: 232-233Crossref PubMed Scopus (231) Google Scholar, 24Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar, 25Parchi P. Zou W. Wang W. Brown P. Capellari S. Ghetti B. Kopp N. Schulz-Schaeffer W.J. Kretzschmar H.A. Head M.W. Ironside J.W. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10168-10172Crossref PubMed Scopus (266) Google Scholar) originally identified two human PrPSc types (named type 1 and type 2). Type 1 has a relative molecular mass of 21 kDa and the primary cleavage site at residue 82, and type 2 has a relative molecular mass of 19 kDa and the primary cleavage at residue 97. In other studies, however, the PrPSc type 1 from codon 129 MM subjects was further distinguished into 2 subtypes showing a less than a 1-kDa difference in mobility (26Wadsworth J.D.F. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar, 27Zanusso G. Farinazzo A. Fiorini M. Gelati M. Castagna A. Righetti P.G. Rizzuto N. Monaco S. J. Biol. Chem. 2001; 276: 40377-40380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). According to Wadsworth et al. (26Wadsworth J.D.F. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar) the two PrPSc type 1 subtypes, they named types 1 and 2, show indistinguishable fragment sizes only when PK digestion is performed in the presence of 20 mm EDTA, thereby representing two distinct conformations acquired by PrPSc in the presence of metal ions such as copper and zinc. In contrast, the two putative PrPSc subtypes described by Zanusso et al. (27Zanusso G. Farinazzo A. Fiorini M. Gelati M. Castagna A. Righetti P.G. Rizzuto N. Monaco S. J. Biol. Chem. 2001; 276: 40377-40380Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), also named types 1 and 2 although not comparable with those of Wadsworth et al. (26Wadsworth J.D.F. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar), would represent two distinct protein conformations because of a different response to pH variations (i.e. one conformation is pH-dependent; the other is not). The rationale for the study of the effect of metal ions and pH on PrPSc properties lies in the finding that the octapeptide repeat PrPc sequence between residues 51 and 91 is a Cu 2+ binding motif that changes its conformation in the presence of copper and that this copper binding stoichiometry is pH-dependent (31Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (248) Google Scholar, 32Kramer M.L. Kratzin H.D. Schmidt B. Römer A. Windl O. Liemann S. Hornemann S. Kretzschmar H. J. Biol. Chem. 2001; 276: 16711-16719Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In previous studies on PrPSc typing in sCJD, we also reported a certain degree of heterogeneity within both type 1 and type 2 samples, particularly among type 1 samples from codon 129 MM subjects (23Parchi P. Capellari S. Chen S.G. Petersen R.B. Gambetti P. Kopp N. Brown P. Kitamoto T. Tateishi J. Giese A. Kretzschmar H. Nature. 1997; 386: 232-233Crossref PubMed Scopus (231) Google Scholar, 24Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar). However, these were subtle biochemical differences compared with the type 1/type 2 difference in relative molecular mass and failed to show a consistent reproducibility or a correlation with the disease pathological phenotype, suggesting that they might be related to the experimental conditions rather than to intrinsic differences in strains. For example, it is noteworthy that the various protocols that have been applied to define PrPSc properties to date included the use of PK well outside its optimal conditions (i.e. pH optimum above 7.5; optimum temperature above 37 °C) (33Ebeling W. Hennrich N. Klockow M. Metz H. Orth H.D. Lang H. Eur. J. Biochem. 1974; 47: 91-97Crossref PubMed Scopus (482) Google Scholar, 34Naureckiene S. Ma L. Sreekumar K. Purandare U. Lo C.F. Huang Y. Chiang L.W. Grenier J.M. Ozenberger B.A. Jacobsen J.S. Kennedy J.D. DiStefano P.S. Wood A. Bingham B. Arch. Biochem. Biophys. 2003; 420: 55-67Crossref PubMed Scopus (132) Google Scholar) and, above all, were not standardized with respect to variables that might affect its activity. On the other hand, the characterization of at least five distinct pathological sCJD subtypes (9Parchi P. Giese A. Capellari S Brown P. Schulz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar) argues for the existence of more than two prion strains and two PrPSc types. Thus, it is conceivable that the application of more sensitive techniques and more rigorous experimental conditions will lead to the distinction between potential artifacts related to sample preparation and disease-specific biochemical differences suitable for a refined classification of CJD PrPSc types. To reach this goal, shed light on the current PrPSc typing controversies, and further contribute to the understanding of the molecular basis of TSE strains and phenotypic variability, we examined the effect of different experimental conditions on the characteristics of PrPSc fragments associated with the sCJD subtypes and vCJD using a high resolution gel electrophoresis system. Particular emphasis has been given to the study of the interplay between the effects of homogenate pH and PK concentration/activity. Patients and Tissues—We studied 75 sCJD cases and 4 vCJD cases phenotypically characterized in regard to clinical and histopathological features, pattern of PrP deposition, PRNP genotype, and Western blot profile of PrPSc. Sporadic CJD subtypes were classified according to Parchi et al. (9Parchi P. Giese A. Capellari S Brown P. Schulz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar). They included 40 MM1, 5 MV1, 10 VV2, 10 MV2, 5 VV1, and 5 MM2-cortical. Brain tissues were obtained at autopsy and were kept frozen at -80 °C until use. Brain samples used were from the frontal cerebral cortex, usually the middle frontal gyrus. In 5 MM1 cases tissue was also obtained from the putamen, entorhinal cortex, hippocampus, and amygdala. Molecular Genetics—Genomic DNA was extracted from blood or frozen brain tissue. Genotyping of the PRNP coding region was performed as described (9Parchi P. Giese A. Capellari S Brown P. Schulz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar). Sample Preparation and pH Measurement—Brain homogenates (10%, w/v) were prepared on ice in the following conditions: 1) lysis buffer (LB) (100 mm NaCl, 10 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mm Tris); 2) lysis buffer with 100 mm Tris (100 mm NaCl, 10 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 100 mm Tris); 3) PBS (Dulbecco's phosphate-buffered saline lacking MgCl2 and CaCl2 (0.2 g/liter KH2PO4, 8.0 g/liter NaCl, 1.15 g/liter Na2HPO4, 0.2 g/liter KCl); 4) PBS with a 10-fold increase in phosphate salt concentration (2.0 g/liter KH2PO4, 8.0 g/liter NaCl, 11.5 g/liter Na2HPO4); 5) PBS with a 10-fold increase in phosphate salt concentration (2.0 g/liter KH2PO4, 8.0 g/liter NaCl, 11.5 g/liter Na2HPO4) and the addition of a detergent (1% Sarkosyl or 1% Nonidet P-40); 6) citrate-phosphate buffer. According to their pKa values, PBS buffers were prepared between pH 5.5 and 8.0, lysis buffers were prepared between at pH 6.7 and 8.0, and the citrate-phosphate buffer was prepared at pH 4.0, 5.0, and 5.5. Because the pH of Tris buffers changes significantly according to the buffer temperature, the lysis buffers were titrated to the desired pH value at 37 °C (i.e. the temperature at which protease digestion is performed). Because sodium deoxycholate is known to precipitate at acidic pH (around 6.3) it was not used at pH values lower than 6.5. Experiments exploring the influence of EDTA on PK digestion were performed using 250 mm EDTA stock solutions at pH 7.0 or 8.0. Samples were treated at 37 °C with PK (20 units/mg, Roche Diagnostics), chymotrypsin (1500 units/mg from bovine pancreas; Calbiochem), or cathepsin L (6500 milliunits/mg, 1.1 mg/ml from bovine kidney; Calbiochem) using various concentration/incubation time combinations (concentration range for PK, 7-10,000 μg/ml; incubation time range for PK, 1-15 h). PK stock solutions (10 mg/ml or higher) were prepared in storage buffer (50% glycerol, 10 mm Tris, pH 7.5, 2.9 mg/ml CaCl2). Small aliquots were prepared and stored at -20 °C. For each experiment a new aliquot was used. Protease digestion was terminated by the addition of 2 mm phenylmethylsulfonyl fluoride. The pH of tissue homogenates was measured in duplicate using a needle electrode (Hamilton). Western Blot—10% brain homogenates were resuspended in sample buffer (final concentration: 3% SDS, 4% β-mercaptoethanol, 10% glycerol, 2 mm EDTA, 62.5 mm Tris, pH 6.8) and boiled for 8 min before loading. Protein samples (brain tissue equivalent to 0.2-1 mg wet tissue) were separated in 12 or 15% SDS-polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) using gel electrophoresis apparatus holding running gels of different lengths (5.5 or 15 cm) (Bio-Rad). Proteins were transferred to Immobilon P (Millipore) for 2 h at 60V, blocked with 10% nonfat milk in Tween Tris-buffered saline, pH 7.5, and probed with the appropriate antibody. The monoclonal 3F4 (1:50,000), which binds PrP between residues 108 and 111 (35Kascsak R.J. Rubenstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar), was used as primary antibody. The immunoreactivity was visualized by enhanced chemiluminescence (Amersham Biosciences) on Kodak BioMax Light films (Eastman Kodak Co.). The Effect of Gel Resolution—To examine the gel mobility of human PrPSc type 1 in more detail than in previous studies we compared the Western blot profiles obtained in 12% Tris-glycine PAGE gels 5.5 cm long (gels commonly used for PrPSc-typing analysis) with those obtained using longer gels (15 cm). In the latter condition, PrPSc extracted from sCJD MM1 showed a more significant heterogeneity. This related either to the number of bands or to the electrophoretic mobility of the most represented bands (Fig. 1, a and b). The Effect of pH—When we compared MM1 samples prepared in LB pH 7.4 with those homogenized in LB pH 8.0 we noticed a higher heterogeneity in gel migration in the first condition (Fig. 1). This observation prompted us to measure the pH of the homogenates. Samples prepared using standard PBS or lysis buffer at pH 7.4 (i.e. buffers that have been used to date for PrPSc-typing studies) showed unexpected heterogeneous pH values ranging from 6.48 to 7.46 (Table I). Similar pH variations were also detected in homogenates prepared from different areas of the same brain (Fig. 2). Homogenates prepared in water were obviously even more acidic (Table I). Interestingly, there was a significant correlation between the pH value of the homogenate and the immunoblot profile of each type 1 sample (Fig. 1). This was also true for samples obtained from different areas of the same brain (Fig. 2). Thus, samples from the same brain may also show a certain degree of heterogeneity in brain homogenate pH as well as electrophoretic mobility of PrPSc core fragments.Table IFrontal cortex homogenate pH values in different buffer conditions (mean ± S.D.)H2OLBLB 100, pH 7.4PBS, pH 7.4pH 7.4pH 8.0MM16.14 ± 0.23 (range 5.86-6.55)6.87 ± 0.21 (range 6.48-7.46)7.80 ± 0.14 (range 7.56-8.04)7.40 ± 0.06 (range 7.36-7.45)6.63 ± 0.23 (range 6.24-6.99)n = 11n = 35n = 17n = 9n = 8sCJD, (all)6.07 ± 0.29 (range 5.43-6.55)6.92 ± 0.19 (range 6.48-7.46)n = 30a11 MM1, 1 MM2, 9 VV2, 9 MV2.n = 69b35 MM1, 3 MM2, 6 VV1, 15 VV2, 10 MV2.a 11 MM1, 1 MM2, 9 VV2, 9 MV2.b 35 MM1, 3 MM2, 6 VV1, 15 VV2, 10 MV2. Open table in a new tab To analyze in detail the effect of the homogenate pH on the characteristics of PrPSc fragments generated by PK digestion, we had to stabilize the pH of the homogenate at the desired value. To this aim, we raised the buffer capacity of the lysis buffer, PBS, and citrate-phosphate buffer solutions and obtained the desired effect by increasing 10-fold either the Tris and the phosphate or citrate-phosphate salt concentration. We found that in sCJD MM1 cases the immunoblot profile of the PrPSc core fragments generated by PK digestion in standard conditions (100 μg/ml at 37 °C for 1 h) varied significantly according to the homogenate pH and showed a shift in gel mobility between pH 6.0 and 8.0 (Fig. 3). At a pH between 6.0 and 6.7 the protein resolved in four distinct fragments. By increasing the pH values of the homogenate we obtained a progressive disappearance of the slowest migrating peptides associated with a parallel increase in the amount of the fastest migrating fragments together with the appearance around pH 7.2 of a novel fragment that migrated even faster. Finally, an additional (6th) slower migrating fragment was detected after digestion at a very acidic pH (5.0-5.5) (Fig. 3), whereas no digestion at all was observed at pH 4.0 (data not shown). Thus, the PrPSc core heterogeneity observed in MM1 subjects using homogenates prepared in standard buffers (LB with 10 mm Tris or standard PBS, pH 7.4), as used in previous studies (9Parchi P. Giese A. Capellari S Brown P. Schulz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1191) Google Scholar, 24Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar, 26Wadsworth J.D.F. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar, 36Collinge J. Sidle K.C.L. Meads J. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1584) Google Scholar), was clearly related to the variability of the homogenate pH (Figs. 1, 2, 3). The Effect of PK Activity—Given that the pH optimum for PK is in the basic pH range, we asked whether the multiple PrPSc fragments generated by protease digestion at acidic pH may be at least in part the consequence of a reduced PK activity. By exposing the samples to different PK concentrations we found that the same heterogeneity among PrPSc fragments generated by PK digestion at various pH values could be obtained by changing the PK concentration at a given pH value. In particular, we observed the appearance of the "ladder effect" on type 1 samples digested at pH 8.0 by progressively decreasing the PK concentration (Fig. 4). Similarly, a complete disappearance of the PrPSc fragments of higher molecular mass that are seen after digestion at acidic pH (6.7) was obtained by increasing the PK concentration 10-15 times (Fig. 4). To determine whether there is also an effect of pH on PrPSc irrespective of its effect on PK activity, we used higher PK concentrations, varying them according to pH to compensate for the change in protease activity and avoid the complete PrPSc digestion. Whereas at pH values below 7.2 PK digestion of PrPSc extracted from sCJD MM1 showed a relative molecular mass of 21 kDa, at pH values higher than 7.3 the protein migrated about 1 kDa faster (Fig. 5). Both fragments were seen at pH 7.2-7.3. All MM1 (n

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