Prion Proteins with Different Conformations Accumulate in Gerstmann-Sträussler-Scheinker Disease Caused by A117V and F198S Mutations
2001; Elsevier BV; Volume: 158; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64692-5
ISSN1525-2191
AutoresPedro Piccardo, Juris J. Liepnieks, Albert William, Stephen R. Dlouhy, Martin R. Farlow, Katherine Young, David Nochlin, Thomas D. Bird, Randal R. Nixon, Melvyn J. Ball, Charles DeCarli, Orso Bugiani, Fabrizio Tagliavini, Merrill D. Benson, Bernardino Ghetti,
Tópico(s)Trace Elements in Health
ResumoGerstmann-Sträussler-Scheinker disease (GSS) is characterized by the accumulation of proteinase K (PK)-resistant prion protein fragments (PrPsc) of ∼7 to 15 kd in the brain. Purified GSS amyloid is composed primarily of ∼7-kd PrP peptides, whose N terminus corresponds to residues W81 and G88 to G90 in patients with the A117V mutation and to residue W81 in patients with the F198S mutation. The aim of this study was to characterize PrP in brain extracts, microsomal preparations, and purified fractions from A117V patients and to determine the N terminus of PrPsc species in both GSS A117V and F198S. In all GSS A117V patients, the ∼7-kd PrPsc fragment isolated from nondigested and PK-digested samples had the major N terminus at residue G88 and G90, respectively. Conversely, in all patients with GSS F198S, an ∼8-kd PrPsc fragment was isolated having the major N terminus start at residue G74. It is possible that a further degradation of this fragment generates the amyloid subunit starting at W81. The finding that patients with GSS A117V and F198S accumulate PrPsc fragments of different size and N-terminal sequence, suggests that these mutations generate two distinct PrP conformers. Gerstmann-Sträussler-Scheinker disease (GSS) is characterized by the accumulation of proteinase K (PK)-resistant prion protein fragments (PrPsc) of ∼7 to 15 kd in the brain. Purified GSS amyloid is composed primarily of ∼7-kd PrP peptides, whose N terminus corresponds to residues W81 and G88 to G90 in patients with the A117V mutation and to residue W81 in patients with the F198S mutation. The aim of this study was to characterize PrP in brain extracts, microsomal preparations, and purified fractions from A117V patients and to determine the N terminus of PrPsc species in both GSS A117V and F198S. In all GSS A117V patients, the ∼7-kd PrPsc fragment isolated from nondigested and PK-digested samples had the major N terminus at residue G88 and G90, respectively. Conversely, in all patients with GSS F198S, an ∼8-kd PrPsc fragment was isolated having the major N terminus start at residue G74. It is possible that a further degradation of this fragment generates the amyloid subunit starting at W81. The finding that patients with GSS A117V and F198S accumulate PrPsc fragments of different size and N-terminal sequence, suggests that these mutations generate two distinct PrP conformers. Prion diseases are neurodegenerative disorders present in both humans and animals.1Prusiner SB Scott MR DeArmond SJ Cohen FE Prion protein biology.Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 2Gambetti P Petersen RB Parchi P Chen SG Capellari S Goldfarb L Gabizon R Montagna P Lugaresi E Piccardo P Ghetti B Inherited prion diseases.in: Prusiner SB Prion Biology and Diseases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1999: 509-583Google Scholar In humans, idiopathic, genetically determined, and transmissible prion disorders have been described.1Prusiner SB Scott MR DeArmond SJ Cohen FE Prion protein biology.Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 2Gambetti P Petersen RB Parchi P Chen SG Capellari S Goldfarb L Gabizon R Montagna P Lugaresi E Piccardo P Ghetti B Inherited prion diseases.in: Prusiner SB Prion Biology and Diseases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1999: 509-583Google Scholar, 3Ghetti B Piccardo P Frangione B Bugiani O Giaccone G Prelli F Dlouhy SR Tagliavini F Prion protein amyloidosis.Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (175) Google Scholar A hypothesized central event in the pathogenesis of these disorders is a conformational change of the normal membrane-associated prion protein (PrPc) into a pathogenic isoform (PrPsc).1Prusiner SB Scott MR DeArmond SJ Cohen FE Prion protein biology.Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar PrPc is detergent soluble, protease sensitive, and has a predominantly α-helical structure. In contrast, PrPsc is insoluble in nondenaturing detergents, is relatively resistant to cleavage by proteinase K (PK), and has an increased β-sheet structure.4Bolton DC McKinley MP Prusiner SB Identification of a protein that purifies with the scrapie prion.Science. 1982; 218: 1309-1311Crossref PubMed Scopus (1023) Google Scholar, 5Meyer RK McKinley MP Bowman KA Braunfeld MB Barry RA Prusiner SB Separation and properties of cellular and scrapie prion proteins.Proc Natl Acad Sci USA. 1986; 83: 2310-2314Crossref PubMed Scopus (519) Google Scholar, 6Caughey BW Dong A Bhat KS Ernst D Hayes SF Caughey WS Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy.Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (750) Google Scholar, 7Safar J Roller PP Gajdusek DC Gibbs Jr, CJ Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein.J Biol Chem. 1993; 268: 20276-20284Abstract Full Text PDF PubMed Google Scholar, 8Pan KM Baldwin M Nguyen J Gasset M Serban A Groth D Mehlhorn I Huang Z Fletterick RJ Cohen FE Prusiner SB Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins.Proc Natl Acad Sci USA. 1993; 90: 10962-10966Crossref PubMed Scopus (2092) Google Scholar The methionine (M)/valine (V) polymorphism at the prion protein gene (PRNP) codon 129 can influence the clinical phenotype produced by a mutation, this is seen most markedly with the D178N mutation, which can cause Creutzfeldt-Jakob disease (CJD) or fatal familial insomnia depending on the presence of M or V at residue 129.2Gambetti P Petersen RB Parchi P Chen SG Capellari S Goldfarb L Gabizon R Montagna P Lugaresi E Piccardo P Ghetti B Inherited prion diseases.in: Prusiner SB Prion Biology and Diseases. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1999: 509-583Google Scholar In CJD, a sporadic or inherited disease characterized by rapid progressive dementia and spongiform degeneration in the cerebrum and cerebellum, two main PrP patterns, PrPsc type 1 and type 2, have been described.9Parchi P Castellani R Capellari S Ghetti B Young K Chen SG Farlow M Dickson DW Sima AA Trojanowski JQ Petersen RB Gambetti P Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease.Ann Neurol. 1996; 39: 767-778Crossref PubMed Scopus (740) Google Scholar, 10Parchi P Wenquan Z Wang W Brown P Capellari S Ghetti B Kopp N Schulz-Schaeffer WJ Kretzschmar H Head MW Ironside J Gambetti P Chen SG Genetic influence on the structural variations of the abnormal prion protein.Proc Natl Acad Sci USA. 2000; 97: 10168-10172Crossref PubMed Scopus (271) Google Scholar PrPsc type 1 after deglycosylation is characterized by a PK-resistant C-terminal fragment of 21 kd, whereas PrPsc type 2 is characterized by a C-terminal protease-resistant peptide of 19 kd.9Parchi P Castellani R Capellari S Ghetti B Young K Chen SG Farlow M Dickson DW Sima AA Trojanowski JQ Petersen RB Gambetti P Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease.Ann Neurol. 1996; 39: 767-778Crossref PubMed Scopus (740) Google Scholar Gerstmann-Sträussler-Scheinker disease (GSS) is a genetically determined autosomal dominant prion disease with a protracted clinical course, clinically characterized by ataxia and cognitive impairment.3Ghetti B Piccardo P Frangione B Bugiani O Giaccone G Prelli F Dlouhy SR Tagliavini F Prion protein amyloidosis.Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (175) Google Scholar GSS is caused by mutations P102L, P105L, A117V, G131V, F198S, D202N, Q212P, and Q217R in PRNP.3Ghetti B Piccardo P Frangione B Bugiani O Giaccone G Prelli F Dlouhy SR Tagliavini F Prion protein amyloidosis.Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (175) Google Scholar, 11Piccardo P Dlouhy SR Lievens PMJ Young K Vinters HV Zimmerman TR Mackenzie IRAM Brown P Gibbs Jr, CJ Gajdusek DC Pocchiari M Bugiani O Ironside J Tagliavini F Ghetti B Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.J Neuropathol Exp Neurol. 1998; 57: 979-988Crossref PubMed Scopus (188) Google Scholar, 12Panegyres PK, Toufexis K, Kakulas BA, Brown P, Ghetti B, Piccardo P, Dlouhy SR, Cervenakova L: A new PRNP mutation (G131V) associated with Gerstmann-Sträussler-Scheinker disease. Arch Neurol (in press)Google Scholar The pathological hallmark of GSS is the accumulation of PrP, with and without amyloid tinctorial properties, in the brain.3Ghetti B Piccardo P Frangione B Bugiani O Giaccone G Prelli F Dlouhy SR Tagliavini F Prion protein amyloidosis.Brain Pathol. 1996; 6: 127-145Crossref PubMed Scopus (175) Google Scholar Our studies have shown that the pattern of PrPsc isoforms in the multiple GSS variants analyzed is different from that seen in CJD.11Piccardo P Dlouhy SR Lievens PMJ Young K Vinters HV Zimmerman TR Mackenzie IRAM Brown P Gibbs Jr, CJ Gajdusek DC Pocchiari M Bugiani O Ironside J Tagliavini F Ghetti B Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.J Neuropathol Exp Neurol. 1998; 57: 979-988Crossref PubMed Scopus (188) Google Scholar, 12Panegyres PK, Toufexis K, Kakulas BA, Brown P, Ghetti B, Piccardo P, Dlouhy SR, Cervenakova L: A new PRNP mutation (G131V) associated with Gerstmann-Sträussler-Scheinker disease. Arch Neurol (in press)Google Scholar, 13Piccardo P Seiler C Dlouhy SR Young K Farlow MR Prelli F Frangione B Bugiani O Tagliavini F Ghetti B Proteinase K resistant prion protein in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).J Neuropathol Exp Neurol. 1996; 55: 1157-1163Crossref PubMed Scopus (51) Google Scholar Patients with CJD present full-length and N-truncated PrP fragments whereas patients with GSS present full-length as well as N- and C-terminal truncated PrP peptides.10Parchi P Wenquan Z Wang W Brown P Capellari S Ghetti B Kopp N Schulz-Schaeffer WJ Kretzschmar H Head MW Ironside J Gambetti P Chen SG Genetic influence on the structural variations of the abnormal prion protein.Proc Natl Acad Sci USA. 2000; 97: 10168-10172Crossref PubMed Scopus (271) Google Scholar, 11Piccardo P Dlouhy SR Lievens PMJ Young K Vinters HV Zimmerman TR Mackenzie IRAM Brown P Gibbs Jr, CJ Gajdusek DC Pocchiari M Bugiani O Ironside J Tagliavini F Ghetti B Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.J Neuropathol Exp Neurol. 1998; 57: 979-988Crossref PubMed Scopus (188) Google Scholar, 12Panegyres PK, Toufexis K, Kakulas BA, Brown P, Ghetti B, Piccardo P, Dlouhy SR, Cervenakova L: A new PRNP mutation (G131V) associated with Gerstmann-Sträussler-Scheinker disease. Arch Neurol (in press)Google Scholar, 13Piccardo P Seiler C Dlouhy SR Young K Farlow MR Prelli F Frangione B Bugiani O Tagliavini F Ghetti B Proteinase K resistant prion protein in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).J Neuropathol Exp Neurol. 1996; 55: 1157-1163Crossref PubMed Scopus (51) Google Scholar, 14Parchi P Chen SG Brown P Zou W Capellari S Budka H Hainfellner J Reyes PF Golden GT Haw JJ Gajdusek DC Gambetti P Different patterns of truncated prion protein fragments correlate with distinct phenotypes in P102L Gerstmann-Sträussler-Scheinker disease.Proc Natl Acad Sci USA. 1998; 95: 8322-8327Crossref PubMed Scopus (186) Google Scholar, 15Chen SG Teplow DB Parchi P Teller JK Gambetti P Autilio-Gambetti L Truncated forms of the human prion protein in normal brain and in prion diseases.J Biol Chem. 1995; 270: 19173-19180Crossref PubMed Scopus (456) Google Scholar Previous studies showed that the amyloid subunit in GSS F198S is a 7-kd peptide with an N terminus at residue G81.16Tagliavini F Prelli F Porro M Rossi G Giaccone G Farlow MR Dlouhy SR Ghetti B Bugiani O Frangione B Amyloid fibrils in Gerstmann-Sträussler-Scheinker disease (Indiana and Swedish kindreds) express only PrP peptides encoded by the mutant allele.Cell. 1994; 79: 695-703Abstract Full Text PDF PubMed Scopus (137) Google Scholar Similar studies in patients from one American and one Alsatian family with GSS A117V revealed that the 7-kd amyloid protein had a major N-terminal cleavage site at residue G81 and G88to G90.17Tagliavini F Prelli F Porro M Rossi G Giaccone G Bird TD Dlouhy SR Young K Piccardo P Ghetti B Bugiani O Frangione B Only mutant PrP participates in amyloid formation in Gerstmann-Sträussler-Scheinker disease with Ala>Val substitution at codon 117.J Neuropathol Exp Neurol. 1995; 54: 416Crossref Google Scholar, 18Tagliavini F Lievens PMJ Tranchant C Warter JM Mohr M Giaccone G Perini F Rossi G Salmona M Piccardo P Ghetti B Beavis RC Bugiani O Frangione B Prelli F A 7 kDa prion protein fragment required for infectivity is the mayor amyloid protein in Gerstmann-Sträussler-Scheinker disease A117V.J Biol Chem. 2001; 276: 6009-6015Crossref PubMed Scopus (120) Google Scholar In addition, we have reported on the presence of PrPsc isoforms of ∼27 to 29, 18 to 19, and 8 kd in brain extracts and microsomal fractions of patients from the Indiana kindred with GSS F198S.13Piccardo P Seiler C Dlouhy SR Young K Farlow MR Prelli F Frangione B Bugiani O Tagliavini F Ghetti B Proteinase K resistant prion protein in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).J Neuropathol Exp Neurol. 1996; 55: 1157-1163Crossref PubMed Scopus (51) Google Scholar In GSS A117V, previous studies have been contradictory in terms of whether or not PK-resistant PrP is present.11Piccardo P Dlouhy SR Lievens PMJ Young K Vinters HV Zimmerman TR Mackenzie IRAM Brown P Gibbs Jr, CJ Gajdusek DC Pocchiari M Bugiani O Ironside J Tagliavini F Ghetti B Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.J Neuropathol Exp Neurol. 1998; 57: 979-988Crossref PubMed Scopus (188) Google Scholar, 19Tateishi J Kitamoto T Doh-ura K Sakaki Y Steinmetz G Tranchant C Warter JM Heldt N Immunochemical, molecular genetic, and transmission studies on a case of Gerstmann-Sträussler-Scheinker syndrome.Neurology. 1990; 40: 1578-1581Crossref PubMed Google Scholar, 20Hedge RS Mastrianni JA Scott MR DeFea KA Tremblay P Torchia M DeArmond S Prusiner SB Lingappa VR A transmembrane form of prion protein in neurodegenerative disease.Science. 1998; 279: 827-834Crossref PubMed Scopus (624) Google Scholar The aims of the present study are to determine the biochemical characteristics of PrP in several patients and an asymptomatic carrier from two unrelated American families with PRNP A117V.21Nochlin D Sumi SM Bird TD Snow AD Leventhal CM Beyreuther K Masters CL Familial dementia with PrP-positive amyloid plaques: a variant of Gerstmann-Sträussler-Scheinker syndrome.Neurology. 1989; 39: 910-918Crossref PubMed Google Scholar, 22Ghetti B Young K Piccardo P Dlouhy SR Pahwa R Lyons KE Koller WC Ma MJ De Carli C Rosenberg RN Gerstmann-Sträussler-Scheinker disease (GSS) with PRNP A117V mutation: neuropathological and molecular studies of a new family.Neuropathol App Neurobiol. 1999; 25: 57Google Scholar We analyzed brain homogenates, microsomal preparations, and purified PrP fractions. In addition, based on the observation that the pattern of digestion of PrPsc depends on the tertiary structure of the protein, we investigated whether PrP conformational isomers are present in phenotypically different GSS variants. To explore this possibility, we determined the N-terminal cleavage sites of the PrP fragments that accumulate in GSS A117V and GSS F198S. The experiments were performed using brain tissue from individuals carrying PRNP mutations A117V and F198S. These individuals (seven patients and one asymptomatic carrier) derive from three families and have been studied genetically and neuropathologically.21Nochlin D Sumi SM Bird TD Snow AD Leventhal CM Beyreuther K Masters CL Familial dementia with PrP-positive amyloid plaques: a variant of Gerstmann-Sträussler-Scheinker syndrome.Neurology. 1989; 39: 910-918Crossref PubMed Google Scholar, 22Ghetti B Young K Piccardo P Dlouhy SR Pahwa R Lyons KE Koller WC Ma MJ De Carli C Rosenberg RN Gerstmann-Sträussler-Scheinker disease (GSS) with PRNP A117V mutation: neuropathological and molecular studies of a new family.Neuropathol App Neurobiol. 1999; 25: 57Google Scholar, 23Ghetti B Tagliavini F Masters CL Beyreuther K Giaccone G Verga L Farlow MR Conneally PM Dlouhy SR Azzarelli B Bugiani O Gerstmann-Sträussler-Scheinker disease. II. Neurofibrillary tangles and plaques with PrP-amyloid coexist in an affected family.Neurology. 1989; 39: 1453-1461Crossref PubMed Google Scholar, 24Farlow MR Yee RD Dlouhy SR Conneally PM Azzarelli B Ghetti B Gerstmann-Straussler-Scheinker disease. I. Extending the clinical spectrum.Neurology. 1989; 39: 1446-1452Crossref PubMed Google Scholar, 25Dlouhy SR Hsiao K Farlow MR Foroud T Conneally PM Johnson P Prusiner SB Hodes ME Ghetti B Linkage of the Indiana kindred of Gerstmann-Sträussler-Scheinker disease to the prion protein gene.Nat Genet. 1992; 1: 64-67Crossref PubMed Scopus (188) Google Scholar, 26Hsiao K Dlouhy S Ghetti B Farlow M Cass C Da Costa M Conneally PM Hodes ME Prusiner SB Mutant prion proteins in Gerstmann-Sträussler-Scheinker disease with neurofibrillary tangles.Nat Genet. 1992; 1: 68-71Crossref PubMed Scopus (217) Google Scholar The data on these cases is summarized in Table 1.Table 1The Clinical, Genetic, and Pathological Information for Five GSS A117V Patients and One Asymptomatic A117V Mutation Carrier; and for Two GSS F198S Patients is SummarizedPrPPatientKADDuration, yearsResidue 129GNeocortexStriatumCerebellumSpongNFT1SG613MVM+/++++−−−2SG393MVF+/+++++/−−−3SG327VVM+/+++/+++/−−−4SG335VVM+/+++++++−−5AS454VVF++/++++++/−−/++−6AS50N/AaMVM−−+/−−−7IK646MVF+++++++++−+++8IK6112VVF+++++++++−+++a, Asymptomatic carrier; K, kindred; AD, age at death; G, gender; Duration, years; duration of clinical signs in years; Spong, spongiform degeneration; NFT, neurofibrillary tangles.Semi-quantitative analysis of lesions: −, absence; +, mild; ++, moderate; +++, severe. Subjects 1 to 6 correspond to two families with GSS A117V and patients 7 and 8 to the Indiana kindred with GSS F198S. Open table in a new tab a, Asymptomatic carrier; K, kindred; AD, age at death; G, gender; Duration, years; duration of clinical signs in years; Spong, spongiform degeneration; NFT, neurofibrillary tangles. Semi-quantitative analysis of lesions: −, absence; +, mild; ++, moderate; +++, severe. Subjects 1 to 6 correspond to two families with GSS A117V and patients 7 and 8 to the Indiana kindred with GSS F198S. Tissue was obtained from six subjects with PRNP A117V. Seven samples from the neocortex and four samples from the cerebellum of each of six individuals were analyzed. Tissue obtained from the frontal cortex of all cases was used for subcellular fractionation, including PrPsc purification and sequencing using Edman chemistry. Tissue was obtained from the frontal cortex and cerebellum of a patient of the Indiana kindred (GSS F198S) heterozygous for the M/V polymorphism at codon 129. In addition, tissues from the frontal cortex, caudate nucleus, and cerebellum of a homozygous V 129 patient from the same kindred were also used for PrPscpurification and amino acid sequencing. Brain tissues from a patient with sporadic CJD and from one individual dying without neurological disease were used as control. Tissue was homogenized in 9 volumes (10% w/v) of lysis buffer (100 mmol/L NaCl, 10 mmol/L ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, 0.5% Na deoxycholate in 10 mmol/L Tris-HCl, pH 7.4). Samples were resolved in 4 to 12% acrylamide gels and transferred to nitrocellulose membranes as described.11Piccardo P Dlouhy SR Lievens PMJ Young K Vinters HV Zimmerman TR Mackenzie IRAM Brown P Gibbs Jr, CJ Gajdusek DC Pocchiari M Bugiani O Ironside J Tagliavini F Ghetti B Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.J Neuropathol Exp Neurol. 1998; 57: 979-988Crossref PubMed Scopus (188) Google Scholar, 13Piccardo P Seiler C Dlouhy SR Young K Farlow MR Prelli F Frangione B Bugiani O Tagliavini F Ghetti B Proteinase K resistant prion protein in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).J Neuropathol Exp Neurol. 1996; 55: 1157-1163Crossref PubMed Scopus (51) Google Scholar Membranes were probed with monoclonal antibody (mAb) 3F4.27Kascsak RJ Rubenstein R Merz PA Tonna-DeMasi M Fersko R Carp R Wisniewski HM Diringer H Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.J Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar Immunoblot analysis for PrPsc was done after digestion of the homogenates with PK (10 μg/ml) for 1 hour at 37°C. Selected samples from patient 3 were also digested with 5, 10, 50, and 100 μg/ml of PK for 1 hour at 37°C. Membrane fractions were obtained as described.13Piccardo P Seiler C Dlouhy SR Young K Farlow MR Prelli F Frangione B Bugiani O Tagliavini F Ghetti B Proteinase K resistant prion protein in Gerstmann-Sträussler-Scheinker disease (Indiana kindred).J Neuropathol Exp Neurol. 1996; 55: 1157-1163Crossref PubMed Scopus (51) Google Scholar, 15Chen SG Teplow DB Parchi P Teller JK Gambetti P Autilio-Gambetti L Truncated forms of the human prion protein in normal brain and in prion diseases.J Biol Chem. 1995; 270: 19173-19180Crossref PubMed Scopus (456) Google Scholar In brief, 10% (w/v) homogenates were prepared in 0.32 mol/L sucrose, 5 mmol/L ethylenediaminetetraacetic acid, 3 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L Tris, pH 7.5. After centrifugation at 6000 ×g for 10 minutes, the pellet (P1) was discarded; the supernatant (S1) was centrifuged at 100,000 × g for 1 hour at 4°C, to obtain a cytosolic fraction (S2) and a membrane fraction (P2). To determine the presence of rough endoplasmic reticulum membranes in these preparations, blots from P2 fractions were probed with mAb 9G10 (Stressgen Biotechnology, Canada) to detect GRP94, a protein normally present in the lumen of the endoplasmic reticulum membrane fraction.28Sastry S Linderoth N Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94).J Biol Chem. 1999; 274: 12023-12035Crossref PubMed Scopus (43) Google Scholar To examine insoluble and PK-resistant PrP in the membrane fraction, the pellet (P2) was resuspended in distilled H2O, incubated on ice for 20 minutes, and centrifuged at 100,000 × g for 1 hour at 4°C. The pellet (P3) was resuspended in 150 mmol/L NaCl, 2% N-lauroylsarcosine (Sarkosyl), 25 mmol/L Tris, pH 7.4, and an aliquot removed for immunoblot analysis. The remaining sample was digested with PK at 100 μg/ml for 1 hour at 37°C. Phenylmethylsulfonyl fluoride was then added to stop PK digestion and the sample was centrifuged at 200,000 × g for 1 hour at 4°C, to obtain insoluble and PK-resistant PrP (P4). The supernatant (S4) was removed from the pellet (P4) and protein present in S4 was precipitated with methanol. The P4 and S4 fractions were resuspended in sample buffer and used for Western blot analysis. PrPsc was purified as described.29Hope J Morton LJD Farquhar CF Multhaup G Beyreuther K Kimberlin R The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).EMBO J. 1986; 5: 2591-2597Crossref PubMed Scopus (261) Google Scholar Tissue (1.5 g) was homogenized (1:4) in 0.01 mol/L Na phosphate, pH 7.4, 10% Sarkosyl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L N-ethylmaleimide, incubated 30 minutes at room temperature and spun at 22,000 ×g for 30 minutes at 10°C. The supernatant (S1) was collected and centrifuged at 215,000 × g for 2.5 hours at 10°C. The pellet (P2) was resuspended in 200 μl of 50 mmol/L Tris-HCl, pH 8.5, and 1 μl of ribonuclease A (80 μg/μl) was added. After stirring for 1 hour at 37°C, 400 μl of solution B (0.6 mol/L K iodide, 6 mmol/L Na thiosulfate, 1% Sarkosyl, 10 mmol/L Na Phosphate, pH 8.5) were added. The sample was centrifuged at 215,000 × g for 1.5 hours at 10°C through a cushion of 20% (w/v) sucrose in solution B (sucrose/sample ratio; 1:4 w/v). The pellet (P3) was resuspended in 110 μl of phosphate-buffered saline. Selected samples were used for immunoblot analysis before PK digestion. Samples from all cases were digested with PK (100 μg/ml for 1 hour at 37°C). For deglycosylation, PrPsc was digested with 5 mU PNGase F (Glyko, Novato, CA) for 2 hours as specified by the manufacturer and analyzed by immunoblot probed with mAb 3F4. In selected samples, polyclonal antibodies AS-6800 raised against synthetic peptides corresponding to residues 89 to 104 (provided by Dr. H. Diringer) and anti PrP-95 to 108 were also used.30Piccardo P Langeveld JPM Hill AF Dlouhy SR Young K Giaccone G Rossi G Bugiani M Bugiani O Meloen RH Collinge J Tagliavini F Ghetti B An antibody raised against a conserved sequence of the prion protein recognizes pathologic isoforms in human and animal prion diseases including vCJD and BSE.Am J Pathol. 1998; 152: 1415-1420PubMed Google Scholar As negative control, filters were probed with the secondary antibody in the absence of primary antibody. An identical protocol was used for the analysis of PrPscisolated from a case of CJD. Purified PrP (ie, non-PK-treated and PK-digested samples) was resolved in 4 to 12% acrylamide gels, transferred to polyvinylidene difluoride Problott membrane (Applied Biosystems, Foster City, CA) and stained with Coomassie blue. The band of interest, as determined by comparison with a Western blot of the same material, was excised and analyzed on an Applied Biosystems model 473A protein sequencer using cycle programs provided by the manufacturer as previously described.31Liepnieks JJ Ghetti B Farlow M Roses AD Benson MD Characterization of amyloid fibril β-peptide in familial Alzheimer's disease with APP 717 mutations.Biochem Biophys Res Comm. 1993; 197: 386-392Crossref PubMed Scopus (11) Google Scholar Immunoblots of brain extracts from subjects carrying the PRNP A117V mutation consistently demonstrated PrP in non-PK-treated homogenates, however considerable heterogeneity of PrPsc was observed. PrPscvaried in overall amount and in the relative quantity of the different isoforms in different brain areas of individual patients and among patients. In many samples obtained from the frontal cortex and cerebellum no PrPsc was detected (data not shown). Nevertheless, PrPsc was seen in selected samples of the frontal cortex of all patients after digestion with PK (10 μg/ml) (Figure 1; lanes B, D, F, H, and J), but not in samples from the asymptomatic carrier (Figure 1, lane L). In cases 1 to 5 (Figure 1; lanes B, D, F, H, and J), a PrPsc band of ∼14 kd was observed, with patients 1, 2, 3, and 5 usually showing an additional band of ∼7 kd (Figure 1; B, D, F, and J). In some blots, a 7-kd band was also seen in patient 4. Extracts obtained from patient 3 showed 7- and 14-kd bands in samples digested with PK at concentrations ranging from 5 to 100 μg/ml (Figure 2). For comparison, we studied in-parallel samples from CJD.Figure 2Immunoblot of PrP from patient 3 after digestion of frontal cortex brain homogenates with 0, 5, 10, 50, and 100 μg/ml of PK (lanes A–E, respectively). Similarly treated samples from a patient with CJD are shown in lanes F–J. Different times of exposure (1 second, 10 seconds, and 30 seconds) were obtained. PrP was detected with mAb 3F4 and visualized by ECL.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PrP partitioned with membranes in samples obtained from the frontal cortex of subjects carrying the PRNP A117V mutation. Examples from two clinically affected and one asymptomatic carrier are shown in Figure 3; lanes A, D, and G. We observed that the membrane fraction obtained from the affected subjects (1 to 5) contained detergent-insoluble and PK-resistant PrP (ie, PrPsc) fragments of 7 and 14 kd. PrPsc present in patients 3 and 4 is shown in Figure 3, lanes B and E. Figure 3 also shows that PrPsc was not detected in the soluble fraction of these patients. Soluble fractions obtained from patients 3 and 4 are shown in Figure 3, lanes C and F. PrPsc was not detected in membranes or in the soluble fractions of the sample obtained from the frontal cortex of the asymptomatic A117V mutation carrier (Figure 3, lanes H and I). To determine the presence of microsomes, immunoblots were probed with mAb 9G10, directed against the heat-shock protein GRP 94 normally present in the lumen of the endoplasmic reticulum membranes. A positive result was obtained (not shown). Because of the variability of detecting abnormal PrP in total brain homogenates and to the fact that previously published protocols are effective in concentrating relatively pure PrPsc, we analyzed partially purified fractions obtained from the frontal cortex of patients 1 to 5 and from the asymptomatic carrier (case 6). After PK digestion, two major bands of 14 and 7 kd were detected in the patients (ie, patients 1 to 5). A similar pattern was seen when immunoblots were probed with a panel of antibodies raised against the mid-region of PrP (ie, PrP residues 89 to 112), however no signal was seen when the primary antibody was omitted. An example of PrPsc banding pattern in patient 5 is shown in Figure 4, lane B. No PrPsc was observed in the partially purified sample obtained from the asymptomatic carrier (not shown). In CJD and some other prion diseases, PrPscis composed of a C-terminal-glycosylated PK-resistant core. The 7-kd
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