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• W2034673903 abstract "The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of α-helical domains accompanied by the increase of β structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of ∼19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation. The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of α-helical domains accompanied by the increase of β structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of ∼19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation. Prion diseases consist of a group of fatal and transmissible neurodegenerative disorders including scrapie and bovine spongiform encephalopathy in animals, and Creutzfeldt-Jakob disease (CJD), 1The abbreviations used are: CJD, Creutzfeldt-Jakob disease; PrPC, the normal, cellular prion protein; PrPSc, the pathogenic and infectious prion protein; GPI, glycosylphosphatidylinositol; PK, proteinase K; IPG, immobilized pH gradient; PrP-CTF12/13, the 12- and 13-kDa PK-resistant C-terminal fragment of PrPSc; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio[-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; PIPLC, phosphatidylinositol-specific phospholipase C; GSS, Gerstmann-Sträussler-Scheinker disease.1The abbreviations used are: CJD, Creutzfeldt-Jakob disease; PrPC, the normal, cellular prion protein; PrPSc, the pathogenic and infectious prion protein; GPI, glycosylphosphatidylinositol; PK, proteinase K; IPG, immobilized pH gradient; PrP-CTF12/13, the 12- and 13-kDa PK-resistant C-terminal fragment of PrPSc; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio[-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; PIPLC, phosphatidylinositol-specific phospholipase C; GSS, Gerstmann-Sträussler-Scheinker disease. fatal familial insomnia (FFI), and Gerstmann-Sträussler-Scheinker disease (GSS) in humans (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5101) Google Scholar, 2Parchi P. Gambetti P. Curr. Opin. Neurol. 1995; 8: 286-293Crossref PubMed Scopus (77) Google Scholar). Human prion diseases can be sporadic, familial, or acquired by infection. Most of them are characterized by the deposition of an abnormal prion protein, PrPSc, in brain. PrPSc derives from its host-encoded normal cellular isoform PrPC that is predominantly expressed in brain but also at lower levels in many other tissues (3Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 4Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 6188-6199Abstract Full Text PDF PubMed Google Scholar, 5Bendheim P.E. Brown H.R Rudelli R.D. Scala L.J. Goller N.L. Wen G.Y. Kascsak R.J. Cashman N.R. Bolton D.C. Neurology. 1992; 42: 149-156Crossref PubMed Google Scholar). Mature human PrPC contains 209 amino acids encompassing residues 23-231, a disulfide bridge between residues 179 and 214, two consensus sites for N-linked glycans at residues 181 and 197, and it is attached to cell membranes via a C-terminal glycosylphosphatidylinositol (GPI) anchor (6Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 7Kretzschmar H.A. Stowring L.E. Westaway D. Stubblebine W.H. Prusiner S.B. DeArmond S.J. DNA. 1986; 5: 315-324Crossref PubMed Scopus (297) Google Scholar, 8Basler K. Oesch B. Scott M. Westaway D. Walchli M. Groth D.F. McKinley M.P. Prusiner S.B. Weissmann C. Cell. 1986; 46: 417-428Abstract Full Text PDF PubMed Scopus (640) Google Scholar, 9Liao Y.C. Lebo R.V. Clawson G.A. Smuckler E.A. Science. 1986; 233: 364-367Crossref PubMed Scopus (110) Google Scholar, 10Turk E. Teplow D.B. Hood L.E. Prusiner S.B. Eur. J. Biochem. 1988; 176: 21-30Crossref PubMed Scopus (269) Google Scholar, 11Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (901) Google Scholar, 12Stahl N. Baldwin M.A. Burlingame A.L. Prusiner S.B. Biochemistry. 1990; 29: 8879-8884Crossref PubMed Scopus (142) Google Scholar). Although PrPC and PrPSc have an identical primary structure, they have distinct physicochemical properties. PrPC exists as a detergent-soluble monomer and is readily degraded by proteinase K (PK), whereas PrPSc forms detergent-insoluble aggregates and shows high resistance to PK digestion (6Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 13Bolton D.C. McKinley M.P. Prusiner S.B. Science. 1982; 218: 1309-1311Crossref PubMed Scopus (1009) Google Scholar, 14Prusiner S.B. McKinley M.P. Bowman K.A. Bolton D.C. Bendheim P.E. Groth D.F. Glenner G.G. Cell. 1983; 35: 349-358Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 15Hope J. Morton L.J. Farquhar C.F. Multhaup G. Beyreuther K. Kimberlin R.H. EMBO J. 1986; 5: 2591-2597Crossref PubMed Scopus (260) Google Scholar, 16Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 17Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (517) Google Scholar). Following treatment with PK, PrPSc generates a protease-resistant core, referred to as PrP27-30 that is N-terminally truncated at the N terminus between residues 78 and 103 (6Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 18Parchi P. Zou W.Q. 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). It is thought that the PrPC to PrPSc conversion involves the decrease of α-helical domains accompanied by the increase of β structures in the midportion of the PrP molecule (19Caughey 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, 20Pan 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, 21Safar J. Roller P.P. Gajdusek D.C. Gibbs C.J. Biochemistry. 1994; 33: 8375-8383Crossref PubMed Scopus (112) Google Scholar). However, the precise location and extent of these structural changes within the PrP molecule and therefore, the tertiary and quaternary structures of PrPSc, are largely a matter of speculation. Full-length PrPSc and PrP27-30 are the only known components of the naturally occurring infectious agent causing prion diseases (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5101) Google Scholar) and are thought to be the primary cause of the histological changes in brains of subjects with prion diseases (22DeArmond S.J. Ironside J.W. Prusiner S.B. Neuropathology of Prion diseases. Prion Biology and Diseases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 585-652Google Scholar). However, the role of PrPSc in the pathogenesis of these changes is poorly understood. This issue is further compounded by the presence of other derivatives of PrPSc in human and animal prion diseases, particularly the 7- 8-kDa-truncated PrP fragments found in GSS (23Tagliavini F. Prelli F. Ghiso J. Bugiani O. Serban D. Prusiner S.B. Farlow M.R. Ghetti B. Frangione B. EMBO J. 1991; 10: 513-519Crossref PubMed Scopus (169) Google Scholar, 24Ghetti B. Piccardo P. Spillantini M.G. Ichimiya Y. Porro M. Perini F. Kitamoto T. Tateishi J. Seiler C. Frangione B. Bugiani O. Giaccone G. Prelli F. Goedert M. Dlouhy S.R. Tagliavini F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 744-748Crossref PubMed Scopus (253) Google Scholar, 25Parchi P. Chen S.G. Brown P. Zou W.Q. Capellari S. Budka H. Hainfellner J. Reyes P.F. Golden G.T. Hauw J.J. Gajdusek D.C. Gambetti P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8322-8327Crossref PubMed Scopus (183) Google Scholar, 26Tagliavini F. Lievens P.M. Tranchant C. Warter J.M. Mohr M. Giaccone G. Perini F. Rossi G. Salmona M. Piccardo P. Ghetti B. Beavis R.C. Bugiani O. Frangione B. Prelli F. J. Biol. Chem. 2001; 276: 6009-6015Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and the 16- and 7-kDa C-terminal fragments in scrapie-infected hamsters (27Kocisko D.A. Lansbury Jr., P.T. Caughey B. Biochemistry. 1996; 35: 13434-13442Crossref PubMed Scopus (86) Google Scholar, 28Caughey B. Raymond G.J. Bessen R.A. J. Biol. Chem. 1998; 273: 32230-32235Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). We have now characterized two novel C-terminal fragments of PrP in brains of patients with sporadic CJD (sCJD). These PrP fragments migrate at about 12 and 13 kDa on Tris-Tricine gradient gel, and have the N terminus that begins at residues 162/167 and 154/156, respectively, as determined by automated Edman degradation. They are resistant to PK digestion and derive from both glycosylated and unglycosylated forms. We propose that these fragments are distinct from PrP27-30 and derive from a subpopulation of full-length as well as N-terminally truncated PrPSc carrying a different conformation. Reagents and Antibodies—Proteinase K (PK) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. Urea, CHAPS, iodoacetamide, dl-dithiothreitol (DTT), and immobilized pH gradient (IPG) strips (pH 3-10, 11 cm long) were from Bio-Rad. Ampholine pH 3-10 and reagents for enhanced chemiluminescence were from Amersham Biosciences. Magnetic beads (Dynabeads M-280, tosyl-activated) were from Dynal Co. (Oslo, Norway). Several well-characterized anti-PrP antibodies, including rabbit anti-C antiserum immunoreactive to human PrP residues 220-231 (29Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), mouse monoclonal antibody 3F4 recognizing an epitope of human PrP residues 109-112 (30Kascsak 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), and mouse monoclonal antibody 8B4 recognizing an epitope between residues 36-43 (31Li R. Liu T. Wong B.S. Pan T. Morillas M. Swietnicki W. O'Rourke K. Gambetti P. Surewicz W.K. Sy M.S. J. Mol. Biol. 2000; 301: 567-573Crossref PubMed Scopus (106) Google Scholar) were used in this study. All other chemicals were purchased from Sigma unless specified otherwise. Brain Tissues—Human brain tissues were collected at autopsy or biopsy and were kept frozen at -80 °C until use. Brains from subjects with sporadic CJD were confirmed by histological examination, and by immunohistochemistry and immunoblotting to show the presence of PrPSc as described (29Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 32Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar). The control human brains were obtained from individuals unaffected by prion disease. Molecular Genetics—Genomic DNA was extracted from frozen brain tissue. The methionine/valine polymorphism at codon 129 was determined as we described previously (33Goldfarb L.G. Petersen R.B. Tabaton M. Brown P. LeBlanc A.C. Montagna P. Cortelli P. Julien J. Vital C. Pendelbury W.W. Haltia M. Wills P.R. Hauw J.J. McKeever P.E. Monari L. Schrank B. Swergold G.D. Autillio-Gambetti L. Gajdusek D.C. Lugaresi E. Gambetti P. Science. 1992; 258: 806-808Crossref PubMed Scopus (597) Google Scholar). PK Digestion and Deglycosylation of PrP—Brain homogenates (10%, w/v) were prepared on ice in lysis buffer (100 mm NaCl, 10 mm EDTA, 0.5% Nonidet P 40, 0.5% sodium deoxycholate, 10 mm Tris-HCl, pH 7.5), and samples were centrifuged at 1,000 × g for 10 min to remove cellular debris. To prepare the detergent-insoluble fraction (P2), brain homogenates were centrifuged at 14,000 × g for 25 min at 4 °C, and supernatants were further centrifuged at 100,000 × g for 1 h at 4 °C. The pellets were resuspended in lysis buffer. For PK digestion of PrP, samples were incubated with PK at 100 μg/ml for 1 h at 37 °C, and the digestion was then terminated by the addition of 3 mm phenylmethylsulfonyl fluoride. For deglycoyslation of the protein, samples were denatured and incubated in the presence of recombinant peptide N-glycosidase F (PNGase F) according to the manufacturer's protocol (Roche Applied Science). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Two-dimensional Gel Electrophoresis, and Immunoblotting— Samples were mixed with an equal volume of 2 × gel loading buffer (6% SDS, 5% β-mercaptoethanol, 4 mm EDTA, 20% glycerol, 125 mm Tris-HCl, pH 6.8) and boiled for 10 min. Proteins were separated by Tris-Tricine SDS-PAGE (10-17% gradient gels, 16 × 20 × 0.1 cm), as described previously (29Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Two-dimensional gel electrophoresis was performed as described by the supplier with minor modifications using PROTEIN IEF cell (Bio-Rad). For the first dimension isoelectric focusing, P2 fraction boiled in gel loading buffer was precipitated by 9-fold volume of chilled acetone at -20 °C for 1.5 h, followed by centrifugation at 16,000 × g for 15 min at 4 °C. The pellets were resuspended in 10% trichloroacetic acid for 2 h at room temperature and were centrifuged at 16,000 × g for 15 min. The pellets were resuspended in 200 μl of rehydration buffer (7 m urea, 2 m thiourea, 2% CHAPS, 0.5% ampholine pH 3-10, and trace amounts of bromphenol blue), and were loaded onto the immobilized pH gradient (IPG) strips for rehydration at 50 V for 14 h. The rehydrated IPG strips were focused for about 40 kVh. For the second dimension SDS-PAGE, the focused IPG strips were equilibrated for 15 min each in equilibration buffer 1 (6 m urea, 2% SDS, 20% glycerol, 2% DTT, and 0.375 m Tris-HCl, pH 8.8), and then in equilibration buffer 2 (6 m urea, 2% SDS, 20% glycerol, 2.5% iodoacetamide and 0.375 m Tris-HCl, pH 8.8). The equilibrated strips were loaded onto 16.5% Tris-Tricine SDS-PAGE gels (Bio-Rad Criterion gels). Electrophoresis was conducted at 120 V. Proteins on the SDS-PAGE gels were electrotransferred onto PVDF membranes at 70 V for 2 h at 4 °C. The membranes were blocked with 5% nonfat milk in TBST (150 mm NaCl, 0.05% Tween 20, 10 mm Tris-HCl, pH 7.6) overnight at 4 °C prior to incubation with antibodies. Membrane-bound proteins were probed with either anti-C antibody at 1:3,000 or with 3F4 antibody at 1:50,000. Following washes with TBST, the PVDF membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (Amersham Biosciences). The PrP bands were visualized on Kodak X-Omat films by enhanced chemiluminescence (ECL Plus kit, Amersham Biosciences). Purification of PK-resistant PrP-CTF 12/13—Brain tissues (5-10 g) were used for purification of PK-resistant PrPSc fragments according to the published method (34Bolton D.C. Bendheim P.E. Marmorstein A.D. Potempska A. Arch. Biochem. Biophys. 1987; 258: 579-590Crossref PubMed Scopus (117) Google Scholar), as modified (35Chen S.G. Parchi P. Brown P. Capellari S. Zou W.Q. Cochran E.J. Vnencak-Jones C.L. Julien J. Vital C. Mikol J. Lugaresi E. Autilio-Gambetti L. Gambetti P. Nat. Med. 1997; 3: 1009-1015Crossref PubMed Scopus (87) Google Scholar, 36Zou W.Q. Colucci M. Gambetti P. Chen S.G. Potter N.T. Characterization of Prion Proteins: Methods in Molecular Biology: Neurogenetics: Methods and Protocols. Humana Press Inc., Totowa, NJ2002: 305-314Google Scholar). After the final sedimentation of PK-resistant PrPSc fragments by ultracentrifugation, the pellets were denatured and treated with PNGase F. PrP-CTF 12/13 were purified further by micropreparative continuous elution SDS-PAGE (16% gel, Mini Prep Cell apparatus, Bio-Rad). Proteins were eluted at a flow rate of 70 μl/min and collected into 400 μl fractions. The fractions containing PrP-CTF 12/13 were pooled and lyophilized. Immunoblotting with anti-C antibody was used to monitor the purification throughout the procedures. N-terminal Protein Sequencing—Purified proteins were separated by 10-20% mini Tris-Tricine SDS-PAGE gradient gels (Novex pre-cast gel, Invitrogen), transferred onto Problott membranes (Applied Biosystems, Foster City, CA), and visualized by Coomassie Blue staining. N-terminal protein sequencing by automated Edman degradation was performed as described previously (18Parchi P. Zou W.Q. 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), at the ProSeq Microsequencing Facility (Boxford, MA) with an Applied Biosystems 477A Protein Sequencer. N-terminal sequencing typically proceeded for 10 cycles. Multiple N-terminal sequences were obtained by alignment of the experimentally determined amino acids at each cycle with the translated human PrP sequence (Swiss-Prot accession number: P04156). Triton X-114 Phase Separation—Phase separation of PrP in Triton X-114 was performed as described (37Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) with minor modification. Brain tissues were homogenized in 1% Triton X-114 in PBS. 200 μl of brain homogenate was centrifuged at 14,000 rpm for 30 s at 4 °C. The supernatant (100 μl) was incubated at 37 °C for 15 min and centrifuged at 14,000 rpm for 1 min. A solution of 1% Triton X-114/PBS (600 μl) was added into the detergent phase and samples were split into two parts. The samples were incubated with (+) or without (-) phosphatidylinositol-specific phospholipase C (PIPLC) (Sigma) at 4 °C overnight, and the phase separation repeated. Sandwich Blotting of GPI-anchored PrP with Proaerolysin—GPI-anchored proteins were detected with proaerolysin-mediated sandwich blotting according to the published procedure (38Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Proteins transferred onto the PVDF membrane were incubated with proaerolysin (Protox Biotech, Victoria, British Columbia, Canada) at 0.5 μg/ml in PBS for 1 h at room temperature. The blot was incubated with anti-proaerolysin monoclonal antibody (Protox Biotech, Victoria, British Columbia, Canada) at 1:8,000 for 1 h. After wash with TBST and incubation with HRP-conjugated IgG (sheep anti-mouse second antibody), the proaerolysin-bound GPI anchored proteins were visualized by enhanced chemiluminescence (ECL Plus kit, Amersham Biosciences). Immunoprecipitation—Immunoprecipitation of PrP from P2 fraction was performed as described (39Zou W.Q. Cashman N.R. J. Biol. Chem. 2002; 277: 43942-43947Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) with minor modifications. In brief, anti-PrP monoclonal antibodies (8B4) at 100 μg were coupled to 7 × 108 tosyl activated magnetic Dynabeads in 1 ml of phosphate-buffered saline, pH 7.5 (PBS) at 37 °C for 20 h, and the beads were then washed twice with 0.1% bovine serum albumin (BSA) in PBS. The antibody-conjugated beads were incubated with 0.1% BSA, 0.2 m Tris-HCl, pH 8.5 at 37 °C for 4 h to block nonspecific binding sites, followed by two washes with 0.1% BSA in PBS. For immunoprecipitation of PrP, 100 μl of antibody-conjugated beads was incubated with 870 μl of lysis buffer in the presence of 30 μl of P2 fraction at room temperature for 3 h. The immune complex-containing beads were washed three times with washing buffer (2% Nonidet P 40 and 2% Tween-20 in PBS, pH 7.5). PK digestion and deglycosylation of PrP were performed as described above. Samples were mixed with 2× gel loading buffer, heated at 95 °C for 5 min, and subjected to SDS-PAGE and immunoblotting. PK-resistant C-terminal Fragments of PrP Are Present in sCJD Brains—We prepared the detergent-insoluble fraction from brains of 29 subjects affected by various subtypes of sCJD, which included five subtypes identified as MM1, VV1, MM2, MV2, and VV2 sCJD, according to the classification of Parchi et al. (40Parchi 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). These preparations were treated with PK and analyzed by Tris-Tricine gradient gels and immunoblotting using anti-C, an antibody to the C-terminal domain of PrP. Following this procedure, we observed two sets of PK-resistant PrP fragments (Fig. 1). The first set consisted of the well-known PrP27-30 (16Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 29Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 32Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar). It migrated as three bands of 30-35, 27-28, and 19-21 kDa that correspond to N-terminally truncated forms of diglycosylated, monoglycosylated, and unglycosylated PrPSc, respectively. Unlike the PrP27-30, the second set of PK-resistant fragments migrated at ∼12 and ∼13 kDa, and were designated PrP C-terminal fragments 12/13 kDa (PrP-CTF 12/13) (Fig. 1). They varied considerably in amount and ratio among different subtypes of sCJD (Table I). In 26 of the 29 sCJD brains examined, PrP-CTF 12/13 accounted for up to 24% of all PK-resistant PrP fragments as measured by densitometry. The analysis of the ratio, which was examined in 13 cases, often showed a better representation of PrP-CTF13 than PrP-CTF12 but the opposite was occasionally true (Table I). The presence, amount and ratio varied from case to case and showed no apparent correlation with the amount of PrP27-30 and the sCJD subtype (Fig. 1 and Table I). In three cases, PrP-CTF 12/13 were present only in trace amounts as their detection required longer exposure of the immunoblots. The study of the brain distribution, carried out in 14 cases showed that PrP-CTF 12/13 generally were better represented in the neocortex than in the hippocampal formation and subcortical structures such as putamen, thalamus, and cerebellum.Table IPrP-CTF12/13 in various subtypes of sporadic CJD Shown are the presence and representation of PrP-CTF12/13 in fifteen cases of sporadic CJD, grouped by the status of polymorphism at codon 129 and type of PK-resistant PrPSc according to the classification of Parchi et al. (32Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (720) Google Scholar, 40Parchi 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). The relative abundance between the 13 and 12 kDa PrP-CTF is qualitatively indicated by + (less abundant) or ++ (more abundant) as estimated by visual inspection of immunoblots probed with anti-C antibody. Due to the closeness of the PrP-CTF12/13 bands, accurate quantitation became difficult and therefore was not attempted. In some cases, only trace amounts of PrP-CTF12/13 were detected following long exposure of immunoblots.CaseCodon 129PrPSc typeFragment (kDa)13121M/M1+++2M/M1+++3M/M1+++4V/V1+++5V/V1+++6V/V1Trace7M/M2+++8M/M2+++9M/M2Trace10V/V2+++11V/V2+++12V/V2Trace13M/V2+++14M/V2+++15M/V2+++ Open table in a new tab PrP-CTF12/13 Are Truncated Glycoforms of PrPSc—Because PrP contains two consensus sites for N-linked glycans at residues 181 and 197 that contributes to its heterogeneity (6Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1246) Google Scholar, 11Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (901) Google Scholar, 12Stahl N. Baldwin M.A. Burlingame A.L. Prusiner S.B. Biochemistry. 1990; 29: 8879-8884Crossref PubMed Scopus (142) Google Scholar), deglycosylation by PNGase F is often used to simplify the gel migration pattern and to reveal the size of the PrP backbone. As shown in Fig. 2, PrP glycoforms were readily detectable in control brains, which upon deglycosylation, shifted mainly to a 27 kDa band corresponding to the full-length PrP and an 18 kDa band corresponding to an N-terminally truncated PrP fragment with an N terminus starting at residue 111/112, as characterized previously (29Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). They were invariably sensitive to PK digestion as expected for normal PrPC. PrPCTF12/13 in the molecular mass range of 12 and 13 kDa were not detected in control brains with or without deglycosylation. PrP-CTF12/13 were barely detectable in untreated brain homogenates from sCJD subjects. However, PK treatment and removal of glycans with PNGase F independently resulted in a substantial increase in amount of PrP-CTF12/13 with the same gel mobility of 12 and 13 kDa as that observed following treatment with both PK and PNGase F (Fig. 2). These findings indicate that PrP-CTF12/13, like PrP27-30 (16Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 29Chen S.G. Teplow D.B. Parchi P. Tell" @default.
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• W2034673903 title "Identification of Novel Proteinase K-resistant C-terminal Fragments of PrP in Creutzfeldt-Jakob Disease" @default.
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