Characterization of Truncated Forms of Abnormal Prion Protein in Creutzfeldt-Jakob Disease
2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês
10.1074/jbc.m801877200
ISSN1083-351X
AutoresSilvio Notari, Rosaria Strammiello, Sabina Capellari, Armin Giese, Maura Cescatti, Jacques Grassi, Bernardino Ghetti, Jan Langeveld, Wen‐Quan Zou, Pierluigi Gambetti, Hans A. Kretzschmar, Piero Parchi,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoIn prion disease, the abnormal conformer of the cellular prion protein, PrPSc, deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrPSc to proteolytic digestion in vitro generates a core fragment of 19–21 kDa, named PrP27–30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27–30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2–3 kDa faster than PrP27–30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27–30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27–30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrPSc C-terminal fragment relatively to the PrP27–30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrPSc or PrP27–30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrPSc in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrPSc and indicate that the characterization of truncated PrPSc forms may further improve molecular typing in CJD. In prion disease, the abnormal conformer of the cellular prion protein, PrPSc, deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrPSc to proteolytic digestion in vitro generates a core fragment of 19–21 kDa, named PrP27–30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27–30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2–3 kDa faster than PrP27–30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27–30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27–30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrPSc C-terminal fragment relatively to the PrP27–30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrPSc or PrP27–30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrPSc in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrPSc and indicate that the characterization of truncated PrPSc forms may further improve molecular typing in CJD. Transmissible spongiform encephalopathies (TSEs), 2The abbreviations used are: TSE, transmissible spongiform encephalopathies; PrP, prion protein; PK, proteinase K; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; vCJD, variant CJD; CTF, C-terminal fragments; PMSF, phenylmethylsulfonyl fluoride; GPI, glycosylphosphatidylinositol; PNGase F, N-glycosidase F; WB, Western blot. 2The abbreviations used are: TSE, transmissible spongiform encephalopathies; PrP, prion protein; PK, proteinase K; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; vCJD, variant CJD; CTF, C-terminal fragments; PMSF, phenylmethylsulfonyl fluoride; GPI, glycosylphosphatidylinositol; PNGase F, N-glycosidase F; WB, Western blot. or prion diseases, are a phenotypically heterogeneous group of neurodegenerative disorders of humans and animals, characterized by abnormal prion protein (PrP) tissue deposits (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. 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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). The two PrPSc types in conjunction with the codon 129 genotype largely explained CJD phenotypic variability and for the first time provided a molecular basis for disease classification (i.e. MM1, MM2, VV1, etc.) (27Parchi 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 (1187) Google Scholar). More recently, the study of PrPSc under stringent pH conditions using a gel electrophoresis technique with increased resolution demonstrated that PrPSc types 1 and 2 are heterogeneous species, which can be further distinguished into six molecular subtypes that better fit the current histopathologic classification of sporadic CJD (sCJD) (28Notari S. Capellari S. Giese A. Westner I. Baruzzi A. Ghetti B. Gambetti P. Kretzschmar H.A. Parchi P. J. Biol. Chem. 2004; 279: 16797-16804Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar).Although for many years full-length PrPSc and its truncated protease-resistant core, PrP27–30, were thought to be the only TSE-specific PrP components, it has been progressively recognized that abnormal PrP comprises additional truncated protein fragments. Unglycosylated PrPSc fragments of 7–8 kDa, truncated at both the N termini and the C termini, were first associated with Gerstmann-Sträussler-Scheinker disease (29Tagliavini F. Prelli F. 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Lansbury Jr., P.T. Caughey B. Biochemistry. 1996; 35: 13434-13442Crossref PubMed Scopus (86) Google Scholar). More recently, two novel C-terminal fragments of PrPSc (PrP-CTF) have been characterized in sCJD (33Zou W.Q. Capellari S. Parchi P. Sy M.S. Gambetti P. Chen S.G. J. Biol. Chem. 2003; 278: 40429-40436Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). These PrPSc fragments have a relative molecular mass of about 12 and 13 kDa (PrP-CTF 12/13), include glycosylated and unglycosylated forms and an intact C terminus, and originate from PrPSc cleavage at residues 162/167 and 154/156, respectively. In one study, the relative amount of PrP-CTF 12/13, although varying considerably from case to case, accounted for between 0 and 25% of the whole PrPSc signal and showed no apparent correlation with the disease subtype (33Zou W.Q. Capellari S. Parchi P. Sy M.S. Gambetti P. Chen S.G. J. Biol. Chem. 2003; 278: 40429-40436Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). At variance, another study (34Satoh K. Muramoto T. Tanaka T. Kitamoto N. Ironside J.W. Nagashima K. Yamada M. Sato T. Mohri S. Kitamoto T. J. Gen. Virol. 2003; 84: 2885-2893Crossref PubMed Scopus (47) Google Scholar) found the PrP-CTF 12/13 (named fPrP 11–12) in iatrogenic CJD without plaques but not in the dura mater-associated iatrogenic CJD with plaques. Two additional putative N-terminally truncated PrPSc fragments, possibly correlating with pathological features of the disease, include a 16–17-kDA unglycosylated fragment found in all PrPSc type 1 sCJD cases and the MM2C sCJD subtype and a fully glycosylated PrP fragment of about 17.5–18 kDa, linked to the sCJD subtypes VV2 and MV2 (35Zanusso G. Farinazzo A. Prelli F. Fiorini M. Gelati M. Ferrari S. Righetti P.G. Rizzuto N. Frangione B. Monaco S. J. Biol. Chem. 2004; 279: 38936-38942Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Lastly, a similar truncated fragment of about 17 kDa was recently detected in sporadic CJDMM2 and variant CJD (vCJD) by another group (36Pan T. Li R. Kang S.C. Pastore M. Wong B.S. Ironside J. Gambetti P. Sy M.S. J. Neurochem. 2005; 92: 132-142Crossref PubMed Scopus (17) Google Scholar).To better characterize these fragments and the novel putative PrP abnormal fragments, we analyzed PrPSc by Western blotting and a panel of antibodies against various PrP epitopes in 40 CJD cases, including all sCJD subtypes and vCJD. We found that abnormal PrP aggregates in CJD include previously undetected PrPSc fragments sharing the primary N-terminal sequence with types 1 and 2 but lacking the very end of the C terminus together with the GPI anchor. Furthermore, we disclosed previously unreported differences in the amount of PrP-CTF 12/13 among CJD subtypes and describe a novel fragment specific to sCJDMM1 generated by PK digestion in partially denaturing conditions.EXPERIMENTAL PROCEDURESPatients and TissuesWe studied 40 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. (27Parchi 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 (1187) Google Scholar) by means of neurohistology, immunohistochemistry, Western blotting, and genetic analyses. Clinical data and relevant medical records were also examined. The sCJD cases included 9 MM1, 4 MV1, 4 VV1, 5 MV2 with kuru plaques, 10 VV2, 3 MM2-cortical (MM2-C), and 5 MM2-thalamic (MM2-T), also known as sporadic fatal insomnia. All the selected cases showed a single PrPSc type in their brain according to the method by Notari et al. (37Notari S. Capellari S. Langeveld J. Giese A. Strammiello R. Gambetti P. Kretzschmar H.A. Parchi P. Lab. Investig. 2007; 87: 1103-1112Crossref PubMed Scopus (50) Google Scholar). Autopsy brain tissues from three subjects free of neurological symptoms and signs and with a negative neuropathologic examination were used as negative controls. Brain tissues were obtained at autopsy and were kept frozen at –80 °C until use. Brain samples used were from the frontal cerebral cortex, striatum, thalamus, midbrain, and cerebellum. In addition, two white matter samples from the frontal lobe and the cerebellum were obtained from three cases of each sCJD group. The study was approved by the local Hospital Ethics Committee.AntibodiesThe following mouse monoclonal antibodies recognizing different human PrP epitopes were used at defined concentrations: 3F4 (residues 108–111) (38Kascsak 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) obtained from Signet Laboratories, at 80 ng/ml, 6H4 (residues 144–152) obtained from Prionics AG, at 200 ng/ml, 12B2 (residues 89–93) (39Langeveld J.P. Jacobs J.G. Erkens J.H. Bossers A. van Zijderveld F.G. van Keulen L.J. BMC Vet. Res. 2006; 2: 19Crossref PubMed Scopus (103) Google Scholar) at 400 ng/ml, and Sha31 (residues 145–152), 12F10 (residues 144–152), SAF60 (residues 157–161), and Pri917 (residues 216–221) (40Feraudet C. Morel N. Simon S. Volland H. Frobert Y. Creminon C. Villette D. Lehmann S. Grassi J. J. Biol. Chem. 2005; 280: 11247-11268Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) at 400 ng/ml. In addition, two rabbit antiserums against either PrP N terminus (residues 23–40) or PrP C terminus (the 2301 antiserum, residues 220–231) were used.Molecular GeneticsGenomic DNA was extracted from blood or frozen brain tissue. Genotyping of the PRNP coding region was performed as described (19Parchi 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).Purification of PrPScBrain tissues (∼5 grams) were used for purification of PK-resistant PrPSc fragments according to a published method (41Bolton D.C. Bendheim P.E. Marmorstein A.D. Potempska A. Arch. Biochem. Biophys. 1987; 258: 579-590Crossref PubMed Scopus (115) Google Scholar), as modified (42Zou W.Q. Colucci M. Gambetti P. Chen S.G. Potter N.T. Neurogenetics: Methods and Protocols (Methods in Molecular Biology). Humana Press Inc., Totowa, NJ2002: 305-314Google Scholar).Analyses of PrPSc Truncated Forms after Removal of GPI AnchorAliquots of PK- and PNGase F-treated brain homogenates were methanol-precipitated, resuspended in 48% aqueous hydrofluoric acid, and incubated at 4 °C for 24 h, as described (43Borchelt D.R. Rogers M. Stahl N. Telling G. Prusiner S.B. Glycobiology. 1993; 3: 319-329Crossref PubMed Scopus (118) Google Scholar).Sample Preparation and Western BlottingBrain homogenates (10%, w/v) were prepared on ice in lysis buffer with high buffer capacity (LB 100) (100 mm Tris, 100 mm NaCl, 10 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate), pH 6.9. Because the pH of Tris buffers changes significantly according to the buffer temperature, the lysis buffers were titrated to pH 6.9 at 37 °C (i.e. the temperature at which protease digestion is performed). Total protein concentration was estimated using a standard colorimetric method based on bicinchoninic acid (Pierce Biotechnology). All samples were diluted to 6 mg of protein/ml before protease digestion. Aliquots were treated for 1 h at 37 °C with PK (Roche Diagnostics, specific activity by certificate of analysis: 47.9 units/mg) at the concentration of 2 units/ml (1 unit corresponds to 50 μg/ml when PK specific activity is 20 units/mg). Protease digestion was terminated by the addition of 2 mm phenylmethylsulfonyl fluoride (PMSF). Samples were diluted 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 of wet tissue) were separated in 13 or 15% SDS-polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) using gel electrophoresis apparatus holding 7-cm running gels (Bio-Rad). Proteins were transferred to Immobilon P (Millipore) for 2 h at 60 V, blocked with 10% nonfat milk in Tween 20-Tris-buffered saline, pH 7.5, and probed with the appropriate antibody. The immunoreactivity was visualized by enhanced chemiluminescence (ECL standard or plus, GE Healthcare) on Kodak BioMax Light films (Eastman Kodak Co.).PK Digestion in Partial Protein Denaturing ConditionsTemperature—Aliquots of brain homogenate (10%) in LB 100, pH 6.9, were treated at different temperatures (37, 50, 60, 70, 80 °C) with 10 units/ml PK for 1 h. Protease digestion was terminated by the addition of 2 mm PMSF. Then samples were resuspended in sample buffer (see above) and boiled for 8 min before loading.SDS Plus Temperature ("PMSF–" Condition)—Brain homogenates (10%) in LB 100, pH 6.9, were treated at 37 °C with 10 units/ml PK for 1 h. Protease digestion was not terminated by the addition of 2 mm PMSF. The samples were resuspended in a β-mercaptoethanol-free sample buffer (final concentration: 3% SDS, 10% glycerol, 2 mm EDTA, 62.5 mm Tris, pH 6.8) and boiled for 8 min. β-mercaptoethanol was added at the final concentration of 4% just before loading.GdnHCl—Aliquots of 50 μl from 10% brain homogenate in phosphate-buffered saline were mixed with 50 μl of GdnHCl stock solutions, with final GdnHCl 1 m. The solution was incubated for 1 h at 37 °C with 10 units/ml PK. The reaction was stopped with 2 mm PMSF. Proteins were precipitated with 8 volumes of methanol for 2 h at –20 °C. Samples were then centrifuged at 15,000 × g for 15 min at 4 °C, and the pellets were resuspended in sample buffer (see above) and boiled for 8 min before loading.RESULTSAnalysis of Truncated PrPSc Forms in CJD, Evidence for Two Novel Fragments—To detect and characterize the full spectrum of truncated PrPSc fragments in CJD, we performed immunoblots on total brain homogenates using a panel of antibodies recognizing different PrP epitopes. In addition to known abnormal PrP truncated species such as PrP27–30 type 1 and PrP-CTF 12/13, the analyses of samples from sCJDMM1, the most common CJD subtype, showed a novel PK-resistant PrP truncated form with an apparent relative molecular mass of 18.5 kDa (Fig. 1A). The fragment was detected by all antibodies tested with epitopes located between PrP residues 89 and 221 (Fig. 1A) except 6H4 (data not shown), which likely recognize a conformational epitope that is not accessible in this fragment. By contrast, the fragment was not seen by antibodies raised against the PrP N terminus (residues 1–70, data not shown) and virtually undetected by the 2301 antiserum, which recognizes an epitope located at the very end (residues 220–231) of the C-terminal moiety (Fig. 1A).The analyses of other sCJD subtypes and vCJD revealed that the 18.5-kDa fragment was only detectable in the MM1/MV1 subtype (Fig. 1B), but a similar faster migrating 17-kDa band was seen in all subtypes associated with PrPSc type 2. The 17-kDa fragment, like the 18.5-kDa band linked to the MM1/MV1 subtype, was readily detected by antibodies with epitopes scattered along PrP residues 99–221 but undetected by the 2301 antiserum (data not shown). At variance with the 18.5-kDa peptide, however, the 17-kDa band was not recognized by 12B2 (Fig. 1C), a monoclonal antibody raised against an epitope (residues 89–93) located between PrPSc types 1 and 2 primary cleavage sites. These results indicate that the 18.5- and 17-kDa fragments have different N-terminal ends, matching those of the PrP27–30 type to which they are associated. In addition, they likely share the C-terminal portion and lack the GPI anchor. To further prove that these fragments lack the GPI anchor, we analyzed in parallel samples treated or not with aqueous hydrofluoric acid, which removes the GPI modification (43Borchelt D.R. Rogers M. Stahl N. Telling G. Prusiner S.B. Glycobiology. 1993; 3: 319-329Crossref PubMed Scopus (118) Google Scholar). As expected, after the GPI loss, PrPSc types 1 and 2 exactly matched the migration of the 18.5- and 17-kDa fragments, whereas no further truncated forms appeared in the 12–16-kDa range (Fig. 1D and data not shown).The intensity of the 17-kDa fragment varied significantly among the type 2-associated CJD subtypes (Fig. 1, B and E). The 17-kDa band showed the highest amounts in vCJD and the lowest in MM2-T. In addition, as a distinctive feature, the 17-kDa band often resolved as a doublet in vCJD (Fig. 1B).As expected from their predicted amino acid sequences based on antibody mapping, indicating that the fragments include the glycosylation sites, the amount of both the 18.5-kDa fragment and the 17-kDa fragment significantly increased after PNGase F treatment (Fig. 2). In this condition, weak traces of the 18.5-kDa band were also seen in the VV1 samples (Fig. 2), indicating that either one of the two fragments actually forms in all CJD subtypes, although in significantly different amounts.FIGURE 2Effect of deglycosylation on the 18.5- and 17-kDa PrPSc fragments. Immunoblot analyses of frontal cortex homogenates from MM1, MM2C, and VV1 subtypes are shown. PK-treated samples were either untreated or treated with PNGase F and probed with SAF60. The 18.5-and 17-kDa fragments are marked with filled and empty arrowheads, respectively. The same result was reproduced twice with samples from at least three subjects for each group. Approximate molecular masses are in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The analyses of partially purified (P3) PrP preparations in sarkosyl showed that the novel fragments copurify with PrPSc and PrP27–30 in the detergent insoluble fraction after extraction in sarkosyl (Fig. 3). The fragment was already visible in the PK-untreated preparations, although it increased in amount after PK treatment.FIGURE 3The 18.5- and 17-kDa fragments are recovered in the P3 fraction. An immunoblot analysis of Sarkosyl extracted PrPSc (P3 fraction) from MM1 and MM2C subjects is shown. The samples were either untreated or treated with PK. Membrane was probed by SAF60. The 18.5- and 17-kDa fragments are marked with filled and empty arrowheads, respectively. The same result was reproduced twice with samples from at least three subjects for each group. Approximate molecular masses are in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of PrP-CTF 12/13 in sCJD Subtypes MV1, VV1, VV2, MV2, MM2-C, and vCJD—In the publication by Zou et al. (33Zou W.Q. Capellari S. Parchi P. Sy M.S. Gambetti P. Chen S.G. J. Biol. Chem. 2003; 278: 40429-40436Abstract Full Text Full Text PDF PubMed Scop
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