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Insoluble Aggregates and Protease-resistant Conformers of Prion Protein in Uninfected Human Brains

2006; Elsevier BV; Volume: 281; Issue: 46 Linguagem: Inglês

10.1074/jbc.m602238200

ISSN

1083-351X

Autores

Jue Yuan, Xiangzhu Xiao, J.E. McGeehan, Zhiqian Dong, Ignazio Calì, Hisashi Fujioka, Qingzhong Kong, G.G. Kneale, Pierluigi Gambetti, Wen‐Quan Zou,

Tópico(s)

Neurological diseases and metabolism

Resumo

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an α-helix to β-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains. Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an α-helix to β-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains. Human prion diseases may be sporadic, familial, or acquired by infection, and include four major phenotypes: Creutzfeldt-Jakob disease (CJD), 3The abbreviations used are: CJD, Creutzfeldt-Jakob disease; PK, proteinase K; PrPC, cellular prion protein; PrPSc, pathological, scrapie isoform of prion protein; PrP*, PrPSc precursor; iPrP, insoluble PrP; g5p, Fd gene 5 protein; NaPTA, sodium phosphotungstate; PrP*20, PK-resistant PrP fragment migrating at ∼20 kDa; PrP*18, PK-resistant PrP fragment migrating at ∼18 kDa; PrP-(27–30), PK-resistant PrP fragment migrating at ∼27–30 kDa; PrP-CTF12/13, PK-resistant PrP C-terminal fragment migrating at ∼12 and 13 kDa; PNGase F, peptide N-glycosidase F; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid; IPG, immobilized pH gradient; PBS, phosphate-buffered saline; non-PrD, non-prion disease; sCJD, sporadic CJD; rPrP, recombinant PrP. Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, and kuru (1Gambetti P. Kong Q. Zou W.Q. Parchi P. Chen S.G. Br. Med. Bull. 2003; 66: 213-239Crossref PubMed Scopus (421) Google Scholar). The central event in the pathogenesis of all forms of prion disease involves a conversion of the host-encoded cellular prion protein PrPC to its pathogenic conformer PrPSc (2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). Whereas PrPC is detergent-soluble and sensitive to proteinase K (PK) digestion, PrPSc forms detergent-insoluble aggregates and is partially resistant to PK (3Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. 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 (1253) Google Scholar, 4Bolton D.C. McKinley M.P. Prusiner S.B. Science. 1982; 218: 1309-1311Crossref PubMed Scopus (1013) Google Scholar, 5Prusiner 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 (832) Google Scholar, 6Hope J. Morton L.J. Farquhar C.F. Multhaup G. Beyreuther K. Kimberlin R.H. EMBO J. 1986; 5: 2591-2597Crossref PubMed Scopus (261) Google Scholar, 7Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (376) Google Scholar). The conversion of PrPC to PrPSc is known to involve a conformational transition of an α-helical to a β-sheet structure of the protein (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, 9Pan 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 (2076) Google Scholar, 10Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. Biochemistry. 1994; 33: 8375-8383Crossref PubMed Scopus (112) Google Scholar) but the in vivo pathway is still poorly understood. Two non-exclusive conversion models have been put forward: refolding (11Griffith J.S. Nature. 1967; 215: 1043-1044Crossref PubMed Scopus (899) Google Scholar, 12Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1748) Google Scholar) and seeding (13Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). In the refolding model, the exogenous PrPSc binds to a PrPC species that has been partially unfolded and the PrPSc-bound PrPC molecule undergoes a refolding process, during which nascent PrPSc is derived from this PrPC species via a conformational transition. The seeding or nucleation-polymerization model proposes that a small amount of abnormal PrPSc or PrPSc precursor (PrP*) is present in the normal brain and is in reversible equilibrium with PrPC. When several precursor monomeric PrP* molecules form a highly ordered nucleus, PrPC can be converted to PrPSc polymers. Clearly, two key elements are required by the seeding model. One is the presence of a small amount of endogenous PrPSc or PrP* in the uninfected brain and the second is the formation of PrPSc-derived oligomers. The seeding model, with the two elements, has been recapitulated in vitro using PrP from various fungal and mammalian sources (14Vanik D.L. Surewicz K.A. Surewicz W.K. Mol. Cell. 2004; 14: 139-145Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 15Jones E.M. Surewicz W.K. Cell. 2005; 121: 63-72Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 16Castilla J. Saa P. Hetz C. Soto C. Cell. 2005; 121: 195-206Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 17Tanaka M. Chien P. Yonekura K. Weissman J.S. Cell. 2005; 121: 49-62Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 18Ross E.D. Minton A. Wickner R.B. Nat. Cell Biol. 2005; 7: 1039-1044Crossref PubMed Scopus (114) Google Scholar). However, this hypothetical endogenous PrPSc, PrP* or "silent prion" has yet to be identified in the uninfected brains (13Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1922) Google Scholar, 19Weissmann C. Nat. Rev. Microbiol. 2004; 2: 861-871Crossref PubMed Scopus (319) Google Scholar, 20Hall D. Edskes H. J. Mol. Biol. 2004; 336: 775-786Crossref PubMed Scopus (73) Google Scholar). We isolated and characterized insoluble PrP aggregates (designated iPrP) from uninfected human brains using sedimentation and size exclusion chromatography. Our data demonstrate that this isoform accounts for about 5–25% of total PrP including full-length and N-terminal truncated forms present in uninfected brains and that it can be captured by Fd gene 5 protein (g5p) and sodium phosphotungstate (NaPTA), two reagents that specifically bind to PrPSc but not to PrPC (21Zou W.Q. Zheng J. Gray D.M. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1380-1385Crossref PubMed Scopus (96) Google Scholar, 22Safar J. Wille H. Itri V. Groth D. Serban H. Torchia M. Cohen F.E. Prusiner S.B. Nat. Med. 1998; 4: 1157-1165Crossref PubMed Scopus (1078) Google Scholar). The state of the various PrP species range from a monomer to small oligomers (less than 200 kDa), to large aggregates (equal to or greater than 2,000 kDa). Moreover, we observed in these brains protease-resistant PrP core fragments migrating at ∼20, 18–19, and ∼7 kDa on immunoblots, after PK digestion and deglycosylation using anti-PrP antibodies. These PK-resistant PrP fragments are derived from the detergent-insoluble fraction (P2) and are present in the fraction captured with g5p. Reagents and Antibodies—NaPTA, PK, and phenylmethylsulfonyl fluoride were purchased from Sigma. Peptide N-glycosidase F (PNGase F) was purchased from New England Biolabs (Beverly, MA) and used following the manufacturer's protocol. Urea, CHAPS, dl-dithiothreitol, iodoacetamide, tributylphosphine, Ampholine pH 3–10, and immobilized pH gradient (IPG) strips (pH 3–10, 11 cm long), and antibody stripping solution were from Bio-Rad (Richmond, CA). Reagents for enhanced chemiluminescence (ECL Plus) were from Amersham Biosciences, Inc. Magnetic beads (Dynabeads M-280, tosyl-activated) were from Dynal Co. (Oslo, Norway). Anti-PrP antibodies, including rabbit antiserum (anti-C) immunoreactive to human PrP residues 220–231 (23Chen 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 (453) Google Scholar), mouse monoclonal antibody 3F4 against human PrP residues 109–112 (24Kascsak 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), mouse monoclonal antibody 1E4 against human PrP-(97–108) (Cell Sciences, Inc., Canton, MA), and 6H4 against human PrP-(144–152) (Prionics AG, Switzerland) were used. Rabbit anti-caveolin-1 polyclonal antibody was from Clontech. Preparation of Gene 5 Protein (g5p)—The g5p was isolated from Escherichia coli TG1(f+) cells following infection with fd bacteriophage. Purification was performed using DNA cellulose affinity and CM-ion exchange chromatography as described (25Oliver A.W. Bogdarina I. Schroeder E. Taylor I.A. Kneale G.G J. Mol. Biol. 2000; 301: 575-584Crossref PubMed Scopus (43) Google Scholar). The purity was >99% as determined by quantitation of Coomassie Blue-stained bands on SDS-PAGE. Brain Tissues—Consent to use autopsy material for research purposes was obtained for all human brain samples. Autopsy was performed within 20 h from death. Biopsy brain tissues were frozen immediately in liquid nitrogen and then transferred to –80 °C for future use. Clinical data and relevant hospital records were examined. The normal human brains were obtained from subjects free of neurological disorders and PrP mutations as indicated by neurohistology, immunohistochemistry, Western blotting, and genetic analysis at the National Prion Disease Pathology Surveillance Center (Cleveland, OH). Six cases including 4 autopsies and 2 biopsies were used and the average age at death was 61 ± 10 years (49–79 years). In addition, 14 sCJD cases were used as controls. The uninfected brain tissues from transgenic mice expressing 2-fold human PrP (26Kong Q. Huang S. Zou W.Q. Vanegas D. Wang M. Wu D. Yuan J. Zheng M. Bai H. Deng H. Chen K. Jenny A.L. O'Rourke K. Belay E.D. Schonberger L.B. Petersen R.B. Sy M.S. Chen S.G. Gambetti P. J. Neurosci. 2005; 25: 7944-7949Crossref PubMed Scopus (206) Google Scholar), hamster, and cow were also used in this study. Preparation of Brain Homogenate and Detergent-soluble (S2) and -Insoluble (P2) Fractions—The 10% (w/v) brain homogenates were prepared in 9 volumes of lysis buffer (10 mm Tris, 150 mm NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 5 mm EDTA, pH 7.4) on ice using pestles with microtubes driven by a cordless motor. When required, brain homogenates were centrifuged at 1,000 × g for 10 min at 4 °C to collect supernatant (S1). To prepare S2 and P2 fractions, S1 were further centrifuged at 35,000 rpm (100,000 × g) in an SW55 rotor (Beckman Coulter, Fullerton, CA) for 1 h at 4°C. After ultracentrifugation, the supernatants that contain the detergent-soluble fraction were transferred into a clean tube. After being washed gently with 1× lysis buffer twice to remove residual supernatant proteins, the pellets that contain detergent-insoluble fraction (P2) were further resuspended in the lysis buffer as described (27Zou 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 (123) Google Scholar). Velocity Sedimentation in Sucrose Step Gradients—Supernatant prepared by centrifugation of 20% brain homogenate at 1,000 × g for 10 min at 4 °C was incubated with an equal volume of 2% Sarcosyl for 30 min on ice. The sample was loaded atop 10–60% step sucrose gradients and centrifuged at 200,000 × g in the SW55 rotor for 1 h at 4°C as described with minor modifications (28Tzaban S. Friedlander G. Schonberger O. Horonchik L. Yedidia Y. Shaked G. Gabizon R. Taraboulos A. Biochemistry. 2002; 41: 12868-12875Crossref PubMed Scopus (183) Google Scholar, 29Pan T. Wong P. Chang B. Li C. Li R. Kang S.C. Wisniewski T. Sy M.S. J. Virol. 2005; 79: 934-943Crossref PubMed Scopus (28) Google Scholar). After centrifugation, the contents of the centrifuge tubes were sequentially removed from the top to the bottom to collect 12 fractions. Aliquots of 12 fractions were subjected to immunoblot analysis as described below. Size Exclusion Chromatography—Superdex 200 HR beads (GE Healthcare) in a 1 × 30-cm column were used to determine the oligomeric state of PrP molecules. Chromatography was performed in an fast protein liquid chromatography system (GE Healthcare) at a flow rate of 0.25 ml/min and fractions of 0.25 ml each were collected as described (28Tzaban S. Friedlander G. Schonberger O. Horonchik L. Yedidia Y. Shaked G. Gabizon R. Taraboulos A. Biochemistry. 2002; 41: 12868-12875Crossref PubMed Scopus (183) Google Scholar). In brief, 200-μl samples, prepared as described above (sucrose step gradients), were injected into the column for each size exclusion run. The molecular weight (Mr) of the various PrP species recovered in different fast protein liquid chromatography fractions was evaluated according to a calibration curve generated with the gel filtration of molecular mass markers (Sigma) including dextran blue (2,000 kDa), thyroglobulin (669 kDa), apoferittin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa). These standards were loaded independently at the concentrations recommended by Sigma in 200-μl sample volumes. The elution volume of blue dextran was used to determine the void volume (V0 = 8.45 ml) and the total volume (Vt = 24 ml) was provided by the product instruction. The peak elution volumes (Ve) were calculated from the chromatogram and fractional retentions. Kav were calculated using the equations: Kav = (Ve–V0)/(Vt–V0) (30Eggington J.M. Haruta N. Wood E.A. Cox M.M. BMC Microbiol. 2004; 4: 1-12Crossref PubMed Scopus (72) Google Scholar). The calibration curve was determined by plotting the Kav of the protein standards against the log Mr of the standards (30Eggington J.M. Haruta N. Wood E.A. Cox M.M. BMC Microbiol. 2004; 4: 1-12Crossref PubMed Scopus (72) Google Scholar). Capture of PrP by g5p—The g5p molecule (100 μg) was conjugated to 7 × 108 tosyl-activated magnetic beads in 1 ml of phosphate-buffered saline (PBS) at 37 °C for 20 h (21Zou W.Q. Zheng J. Gray D.M. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1380-1385Crossref PubMed Scopus (96) Google Scholar). The g5p-conjugated beads were incubated with 0.1% bovine serum albumin in PBS to block nonspecific binding. The prepared g5p beads were stable for at least 3 months at 4 °C. The capture of PrP by g5p was performed as described (21Zou W.Q. Zheng J. Gray D.M. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1380-1385Crossref PubMed Scopus (96) Google Scholar) incubating S1 fractions or P2 with g5p-conjugated beads (10 μg of protein/6 × 107 beads) in 1 ml of binding buffer (3% Tween 20, 3% Nonidet P-40 in PBS, pH 7.5). After incubation with constant rotation for 3 h at room temperature, the PrP-containing g5p beads were attracted to the sidewall of Eppendorf tubes by external magnetic force, allowing easy removal of all unbound molecules in the solution. Following three washes in wash buffer (2% Tween 20 and 2% Nonidet P-40 in PBS, pH 7.5), the g5p beads were collected and heated at 95 °C for 5 min in SDS sample buffer (3% SDS, 2 mm EDTA, 10% glycerol, 50 mm Tris-HCl, pH 6.8). Precipitation of PrP by NaPTA—Precipitation of PrP by NaPTA was conducted as described (31Wadsworth J.D. Joiner S. Hill A.F. Campbell T.A. Desbruslais M. Luthert P.J. Collinge J. Lancet. 2001; 358: 171-180Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar) with minor modifications. Briefly, 10% (w/v) homogenates from brain tissues were prepared in PBS lacking Ca2+ and Mg2+. The samples were centrifuged at 1,000 × g for 10 min at 4 °C. A 500-μl aliquot of supernatant was mixed with an equal volume of 4% (w/v) Sarcosyl prepared in PBS, pH 7.4, and incubated for 10 min at 37 °C with constant agitation. Samples were adjusted to final concentrations of 50 units/ml Benzonase (Benzon nuclease, Merck & Co, Whitehouse Station, NJ) and 1 mm MgCl2 and incubated for 30 min at 37 °C with constant agitation. Subsequently, the samples were adjusted to a final concentration in the sample of 0.3% (w/v) NaPTA with 81.3 μl of a stock solution containing 4% (w/v) NaPTA and 170 mm MgCl2. Samples were incubated at 37 °C for 30 min with constant agitation before centrifugation at 16,000 × g for 30 min. After removal of the supernatant, the pellet was resuspended in 1× lysis buffer for Western blotting as described below. One- and Two-dimensional Gel Electrophoresis and Immunoblotting—Samples were resolved either on 15% Tris-HCl Criterion pre-cast gels (Bio-Rad) for one-dimensional gel electrophoresis or IPG strips for the two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis was performed as described by the supplier using the PROTEIN IEF cell (Bio-Rad) (27Zou 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 (123) Google Scholar, 32Pastore M. Chin S.S. Bell K.L. Dong Z. Yang Q. Yang L. Yuan J. Chen S.G. Gambetti P. Zou W.Q. Am. J. Pathol. 2005; 167: 1729-1738Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Samples denatured by boiling in SDS sample buffer were incubated with reducing buffer (8 m urea, 2% CHAPS, 5 mm tributylphosphine, 20 mm Tris, pH 8.0) for 1 h at room temperature and then incubated with 200 mm iodoacetamide for 1 h. Proteins were precipitated with a 5-fold volume of pre-chilled methanol at –20 °C for 2 h and centrifuged at 16,000 × g for 20 min at 4 °C. The pellets were resuspended in 200 μl of rehydration buffer (7 m urea, 2 m thiourea, 1% dithiothreitol, 1% CHAPS, 1% Triton X-100, 1% Ampholine pH 3–10, and trace amounts of bromphenol blue). The pellets were dissolved in rehydration buffer and subsequently incubated with the IPG strips for 14 h at room temperature with gentle shaking. The dehydrated gel strips were transferred onto a focusing tray and focused for about 40 kV·h. The focused IPG strips were equilibrated for 15 min in equilibration buffer 1 (6 m urea, 2% SDS, 20% glycerol, 130 mm dithiothreitol, 375 mm Tris-HCl, pH 8.8), and then another 15 min in equilibration buffer 2 (6 m urea, 2% SDS, 20% glycerol, 135 mm iodoacetamide, 375 mm Tris-HCl, pH 8.8). The equilibrated strips were loaded onto the 8–16% Tris-HCl Criterion pre-cast gel (Bio-Rad). The proteins on the gels were transferred to Immobilon-P membrane polyvinylidene fluoride (Millipore) for 2 h at 70 V. For probing of the PrP or caveolin-1, the membranes were incubated for 2 h at room temperature with anti-PrP antibodies including 3F4 (1:40,000), 1E4 (1:500), anti-C (1:4,000), 6H4 (1:10,000), or anti-caveolin-1 (1:5,000) as the primary antibody. Following incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG or donkey anti-rabbit IgG at 1:3,000, the PrP or caveolin-1 bands or spots were visualized on Kodak film by ECL Plus as described by the manufacturer. Detergent-insoluble PrP Species Are Present in Uninfected Human Brains—It has been shown that PrPC is recovered in a soluble fraction (S2), following ultracentrifugation in nondenaturing detergents at 100,000 × g for 1 h at 4 °C, whereas PrPSc is recovered in an insoluble fraction (P2) (6Hope J. Morton L.J. Farquhar C.F. Multhaup G. Beyreuther K. Kimberlin R.H. EMBO J. 1986; 5: 2591-2597Crossref PubMed Scopus (261) Google Scholar, 7Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 23Chen 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 (453) Google Scholar). When we applied this separation procedure to human brain tissues, free of prion diseases (non-PrD), as expected, most of the PrPC was recovered in the S2 fraction (Fig. 1, A and B). Surprisingly, a small amount of PrP was consistently detectable in the P2 fraction (Fig. 1, A and B). Quantitative densitometric analysis of immunoblots probed with 3F4 antibody showed that 96% of the PrP detected was recovered in S2 as a soluble form, whereas 4% of the PrP was recovered in P2 as a detergent-insoluble form (mean ± S.D.: 96 ± 2 versus 4 ± 2%, n = 6) (Fig. 1A). An increased intensity of PrP staining was observed in P2 on the blot re-probed with anti-C (mean ± S.D.: 75 ± 9 (S2) versus 25 ± 9% (P2), n = 6) (Fig. 1B), suggesting that predominant PrP distributed in P2 is N-terminal truncated. Taken together, PrP recovered in P2 accounted for ∼5–25% of total PrP including full-length and N-terminal-truncated species. This novel isoform, termed insoluble PrP (iPrP), had a banding profile different from that of soluble PrPC in the immunoblot probed with 3F4 (Fig. 1, A and C). In normal human brains, PrPC from S2 always possesses a dominant upper band. The intensity of the middle PrP band is often similar or less than that of the upper band. However, the intensity of its low band normally is the lowest among the three PrP bands (Fig. 1C, middle panel). By contrast, the ratio of the low to upper PrP band of the iPrP species significantly increased, which is similar to that found with PrPSc (1Gambetti P. Kong Q. Zou W.Q. Parchi P. Chen S.G. Br. Med. Bull. 2003; 66: 213-239Crossref PubMed Scopus (421) Google Scholar) (Fig. 1C, left and right panels). Following PK treatment, the PK-resistant core fragment (termed PrP-(27–30)) was only detected with 3F4 in PrPSc, but not in either iPrP or PrPC (Fig. 1C). Uninfected Human Brains Contain PrP Aggregates That Are Typically Detected in Prion-infected Brains—It is conceivable that the iPrP molecules also form aggregates in uninfected human brains. To investigate this possibility, velocity sedimentation of PrP in sucrose step gradients from 10 to 60% was conducted. Various PrP species including PK-sensitive aggregates of heterogeneous sizes can be separated by this procedure into several fractions based upon distinct molecular densities, sizes, and shapes (28Tzaban S. Friedlander G. Schonberger O. Horonchik L. Yedidia Y. Shaked G. Gabizon R. Taraboulos A. Biochemistry. 2002; 41: 12868-12875Crossref PubMed Scopus (183) Google Scholar, 29Pan T. Wong P. Chang B. Li C. Li R. Kang S.C. Wisniewski T. Sy M.S. J. Virol. 2005; 79: 934-943Crossref PubMed Scopus (28) Google Scholar). The abundant PrP from the brains of non-PrD was distributed through the upper fractions from 1 to 4 (Fig. 2A). By contrast, PrP from brains infected with prion disease was predominantly recovered in the bottom fractions from 9 to 12 (Fig. 2B). The distribution profile of PrP from non-PrD and sCJD was significantly different. The majority of PrP species recovered in the top fractions from non-PrD represent PrPC that consisted mainly of detergent-soluble monomers. The increased PrP in fractions 9–12 from sCJD indicate that PrPSc formed large aggregates that fractionated toward the bottom of the sucrose gradients (28Tzaban S. Friedlander G. Schonberger O. Horonchik L. Yedidia Y. Shaked G. Gabizon R. Taraboulos A. Biochemistry. 2002; 41: 12868-12875Crossref PubMed Scopus (183) Google Scholar, 29Pan T. Wong P. Chang B. Li C. Li R. Kang S.C. Wisniewski T. Sy M.S. J. Virol. 2005; 79: 934-943Crossref PubMed Scopus (28) Google Scholar). However, small amounts of PrP were also consistently detectable in the bottom fractions derived from the brains of non-PrD. The amount of PrP aggregates precipitating in bottom fractions 9–12 (Fig. 2, A and C) accounted for ∼5% of the total PrP in non-PrD (n = 6), similar to the amount of iPrP detected with 3F4 in fraction P2 (Fig. 1). By contrast, the PrPSc aggregates in fractions 9–12 from sCJD brain homogenates (n = 4) accounted for ∼50.4% of the total PrP (Fig. 2, B and C). The high reproducibility of finding of PrP aggregates in 6 non-PrD brains including 2 biopsy brain samples makes it unlikely that they are a product of postmortem autolysis. We then conducted a comparison of PrP levels in the top and bottom fractions between non-PrD and sCJD on a single blot, which should determine more precisely the changes in the exact amounts of the PrP species in these fractions. Equal volumes of samples from either the top (fraction 1), or the bottom (fraction 9), of a non-PrD and two sCJD cases, were each loaded into the same gel. Compared with non-PrD, the level and/or banding pattern of PrP in fraction 1 (top) for sCJD was profoundly different, in addition to a significant increase in the amount of PrP in fraction 9 (bottom) (Fig. 2D). Therefore, in the brains with non-PrD, whereas most PrP species consisting of monomeric PrPC were recovered in the top fractions of sucrose gradients, there were also small amounts of PrP recovered in the bottom fractions. Because these PrP species were present in all bottom fractions, the density and size must have increased continuously, which is an indication of aggregation of the PrP (28Tzaban S. Friedlander G. Schonberger O. Horonchik L. Yedidia Y. Shaked G. Gabizon R. Taraboulos A. Biochemistry. 2002; 41: 12868-12875Crossref PubMed Scopus (183) Google Scholar, 29Pan T. Wong P. Chang B. Li C. Li R. Kang S.C. Wisniewski T. Sy M.S. J. Virol. 2005; 79: 934-943Crossref PubMed Scopus (28) Google Scholar). By contrast, in the sCJD-affected brains, the PrP species in the bottom fractions significantly increased, whereas PrP in the top fractions decreased, indicating that most of the PrP species was in aggregate form, along with a decrease in the level of PrPC, a result consistent with our recent study on characterization of PrP in the most common subtype of sCJD (33Cali I. Capellari S. Yuan J. Al-Shekhlee A. Cohen M.L. Xiao X. Moleres F.J. Parchi P. Zou W.Q. Gambetti P. Brain. 2006; 129: 2266-2277Crossref PubMed Scopus (108) Google Scholar). In addition, we also investigated hamsters and cows to see if the finding of the presence of iPrP in uninfected human brains is seen in other animals. A small amount of PrP was also detectable in the sucrose gradient bottom fractions of uninfected cow and hamster brains (Fig. 2, E and F). We next examined another membrane protein caveolin-1, as a control, to confirm that this assay is able to faithfully determine the oligomeric state of a membrane protein. Caveolin-1 is mainly localized in plasma-membrane caveolae and may also have soluble cytoplasmic and secreted forms (34Williams T.M. Lisanti M.P. Ann. Med. 2004; 36: 584-595Crossref PubMed Scopus (313) Google Scholar). It has been demonstrated that both recombinant and endogenous caveolin-1 form high molecular mass oligomers of 200–400 kDa in vitro and in vivo (35Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 36Sargiacomo M. Scherer P.E. Tang Z. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (478) Google Scholar). After stripping 3F4 from used Western blots, we re-probed the blots with rabbit anti-caveolin-1 polyclonal antibody. Caveolin-1 from both uninfected and prioninfected human brains was mostly distributed in fractions 2–6, although it was also observed in other fractions (Fig. 3, A and B). Compared with non-PrD, sCJD had higher levels of caveolin-1 in fractions 2–6. Therefore, in the uninfected human brains the membrane protein caveolin-1 is mainly present as small oligomers and small amounts of large aggregates are also detectable, which is different from the membrane protein PrPC. Prion-infected human brain showed an increase in the amounts of only oligomeric caveolin-1 but not large caveolin-1 aggregates. This is inconsistent with the distribution of PrP. The Size of iPrP Aggregates in Uninfected Human Brains Is Similar to That of PrPSc Aggregates Present in Prion-infected Brains—The size of the various PrP conformers of non-PrD samples was further characterized using size exclusion chromatography (also called gel filtration). We first generated a calibration curve with seven molecular mass markers from Sigma (Fig. 4A). We then examined the Mr of PrP from non-PrD and sCJD. PrP fr

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