FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2018542940> ?p ?o ?g. }

• W2018542940 endingPage "15545" @default.
• W2018542940 startingPage "15536" @default.
• W2018542940 abstract "The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159–174 and 224–230 were shown to be buried and were the most resistant to denaturation. The 132–156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95–105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89–112 and 136–158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils. The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159–174 and 224–230 were shown to be buried and were the most resistant to denaturation. The 132–156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95–105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89–112 and 136–158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils. Misfolding and aggregation of the prion protein (PrP) 4The abbreviations used are: PrP, prion protein; rPrP, full-length recombinant prion protein; PrPC, cellular isoform of the prion protein; PrPSc, disease-associated isoform of the prion protein; CJD, Creutzfeldt-Jakob disease; PK, proteinase K; GdnHCl, guanidine hydrochloride; Ab, antibody; TBS, Tris-buffered saline; 2D-FIS, two-dimensional fluorescence intensity scattering plot; LRS, linear regression slopes.4The abbreviations used are: PrP, prion protein; rPrP, full-length recombinant prion protein; PrPC, cellular isoform of the prion protein; PrPSc, disease-associated isoform of the prion protein; CJD, Creutzfeldt-Jakob disease; PK, proteinase K; GdnHCl, guanidine hydrochloride; Ab, antibody; TBS, Tris-buffered saline; 2D-FIS, two-dimensional fluorescence intensity scattering plot; LRS, linear regression slopes. has been linked to several fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Sheinker disease, and fatal familial insomnia (1Prusiner S.B. N. Engl. J. Med. 2001; 344: 1516-1526Crossref PubMed Scopus (665) Google Scholar). Prion maladies manifest themselves in sporadic, familial, or infectious forms (2Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (851) Google Scholar). These diseases, including sporadic CJD, display substantial variations in clinical symptoms, in neuropathological profile, and in age at onset of disease (3Hill A.F. Joiner S. Wadsworth J.D.F. Sidle K.C.L. Bell J.E. Budka H. Ironside J.W. Collinge J. Brain. 2003; 126: 1333-1346Crossref PubMed Scopus (267) Google Scholar, 4Parchi 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 (1172) Google Scholar). This broad pathological and clinical heterogeneity is believed to be related, at least in part, to conformational variations in the disease-related isoforms of PrP (PrPSc). Numerous studies have shown that different strains of transmissible spongiform encephalopathy are also linked to conformational differences within the PrPSc isoform (5Bessen R.A. Marsh R.F. J. Virol. 1994; 68: 7859-7868Crossref PubMed Google Scholar, 6Telling G.C. Parchi P. DeArmond S.J. cortelli P. Montagna P. Gabizon R. Mastrianni J. Lugaresi E. Gambetti P. Prusiner S.B. Science. 1996; 274: 2079-2082Crossref PubMed Scopus (741) Google Scholar, 7Safar 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 (1064) Google Scholar, 8Peretz D. Scott M. Groth D. Williamson A. Burton D. Cohen F.E. Prusiner S.B. Protein Sci. 2001; 10: 854-863Crossref PubMed Scopus (211) Google Scholar, 9Caughey B. Raymond G.J. Bessen R.A. J. Biol. Chem. 1998; 273: 32230-32235Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 10Thomzig A. Spassov S. Friedrich M. Naumann D. Beekes M. J. Biol. Chem. 2004; 27933854Abstract Full Text Full Text PDF Scopus (73) Google Scholar). Coexistence of multiple types of PrPSc was recently shown in patients with sporadic CJD (11Polymenidou M. Stoeck K. Glatzel M. Vey M. Bellon A. Aguzzi A. Lancet Neurol. 2005; 4: 805-814Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Schoch G. Seeger H. Bogousslavsky J. Tolnay M. Janzer R.C. Aguzzi A. Glatzel M. PLoS Med. 2006; 3: 236-244Crossref Scopus (93) Google Scholar) and in variant CJD (13Yull H.M. Ritchie D.L. Langeveld J.P. van Zijderveld F.G. Bruce M.E. Ironside J.W. Head M.W. Am. J. Pathol. 2006; 168: 151-157Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The coincidence of multiple prion strains or subtypes in natural prion isolates demands the development of an assay that is able to assess the conformational heterogeneity in mixtures of abnormal PrP isoforms. Over the past decade, a variety of biochemical and immunological assays have been established that discriminate PrPSc from PrPC and distinguish different strains or subtypes of PrPSc. These assays recognize different conformers of PrPSc by the extent of PK resistance, the size of the PK-resistant core, thermodynamic stability, or epitope presentation (7Safar 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 (1064) Google Scholar, 8Peretz D. Scott M. Groth D. Williamson A. Burton D. Cohen F.E. Prusiner S.B. Protein Sci. 2001; 10: 854-863Crossref PubMed Scopus (211) Google Scholar, 14Peretz D. Williamson R.A. Matsunaga Y. Serban H. Pinilla C. Bastidas R.B. Rozenshteyn R. James T.L. Houghten R.A. Cohen F.E. Prusiner S.B. Burton D.R. J. Mol. Biol. 1997; 273: 614-622Crossref PubMed Scopus (319) Google Scholar, 15Paramithiotis E. Pinard M. Lawton T. LaBoissiere S. Leathers V.L. Zou W.Q. Estey L.A. Lamontagne J. Lehto M.T. Kondejewski L.H. Francoeur G.P. Papadopoulos M. Haghighat A. Spatz S.J. Head M. Will R. Ironside J. O'Rouke K. Tonelli Q. Ledebur H.C. Chakrabartty A. Cashman N.R. Nat. Med. 2003; 9: 893-899Crossref PubMed Scopus (240) Google Scholar, 16Bessen R.A. Marsh R.F. J. Virol. 1992; 66: 2096-2101Crossref PubMed Google Scholar, 17Kuczius T. Groschup M.H. Mol. Med. 1999; 5: 406-418Crossref PubMed Google Scholar, 18Bellon A. Seyfert-Brandt W. Lang W. Baron H. Groner A. Vey M. J. Gen. Virol. 2003; 84: 1921-1925Crossref PubMed Scopus (84) Google Scholar). All previously developed assays, however, assess bulk properties of PrP molecules averaged across the whole molecular population but not the conformation of individual fibrils or particles. When multiple strains or subtypes of PrPSc are present as a mixture, analysis of such samples is difficult and often produces conflicting results (3Hill A.F. Joiner S. Wadsworth J.D.F. Sidle K.C.L. Bell J.E. Budka H. Ironside J.W. Collinge J. Brain. 2003; 126: 1333-1346Crossref PubMed Scopus (267) Google Scholar, 4Parchi 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 (1172) Google Scholar, 12Schoch G. Seeger H. Bogousslavsky J. Tolnay M. Janzer R.C. Aguzzi A. Glatzel M. PLoS Med. 2006; 3: 236-244Crossref Scopus (93) Google Scholar, 19Notari 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 (121) Google Scholar). Similar to the high heterogeneity of PrPSc subtypes generated in sporadic CJD, spontaneous polymerization of PrP in vitro under single growth conditions produces a range of amyloid fibrillar types (20Anderson M. Bocharova O.V. Makarava N. Breydo L. Salnikov V.V. Baskakov I.V. J. Mol. Biol. February 20, 2006; 10.1016/j.jmb.2006.02.007Google Scholar). The specific conformational differences between fibrillar types have not yet been determined. Elucidation of the physical properties and conformational heterogeneity of individual synthetic fibrils is very important, considering that only a very small subfraction of fibrils produced in vitro appear to be infectious (21Legname G. Baskakov I.V. Nguyen H.-O.B. Riesner D. Cohen F.E. DeArmond S.J. Prusiner S.B. Science. 2004; 305: 673-676Crossref PubMed Scopus (893) Google Scholar). Although the fibrillar form of recombinant PrP was shown to induce transmissible prion disease in animals (21Legname G. Baskakov I.V. Nguyen H.-O.B. Riesner D. Cohen F.E. DeArmond S.J. Prusiner S.B. Science. 2004; 305: 673-676Crossref PubMed Scopus (893) Google Scholar, 22Legname G. Nguyen H.-O.B. Baskakov I.V. Cohen F.E. DeArmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2168-2173Crossref PubMed Scopus (161) Google Scholar), our current knowledge about the specific structural features that underlie prion infectivity is very limited. In recent studies, solid state NMR was used to determine the three-dimensional structure of fibrils produced from several amyloidogenic polypeptides, including Aβ(1–42) (23Petkova A.T. Leapman R.D. Gua Z. Yau W.-M. Mattson M.P. Tycko R. Science. 2005; 307: 262-265Crossref PubMed Scopus (1401) Google Scholar, 24Luhrs T. Ritter C. Adrian M. Riek-Loher D. Bohrmann B. Dobeli H. Schubert D. Riek R. Proc. Acad. Natl. Sci. U. S. A. 2005; 102: 17342-17347Crossref PubMed Scopus (1654) Google Scholar), yeast prion Ure2p peptide 10–39 (25Chan J.C.C. Oyler N.A. Yau W.M. Tycko R. Biochemistry. 2005; 44: 10669-10680Crossref PubMed Scopus (125) Google Scholar), and fungal prion HET-s peptide 218–289 (26Ritter C. Maddelein M.L. Siemer A.B. Luhrs T. Ernst M. Meier B.H. Saupe S.J. Riek R. Nature. 2005; 435: 844-848Crossref PubMed Scopus (395) Google Scholar). To date, the solid state NMR studies have been restricted to relatively short polypeptides that produce conformationally homogeneous well defined fibrils. The large size of full-length PrP molecules in combination with the highly heterogeneous nature of PrP fibrils limits the number of physical techniques that can be used for assessing the conformation of aggregated states of PrP. Antibodies have previously proved to be an informative tool for monitoring the structural transition from PrPC to PrPSc and for probing the conformation of aggregated disease-specific isoforms of PrP (14Peretz D. Williamson R.A. Matsunaga Y. Serban H. Pinilla C. Bastidas R.B. Rozenshteyn R. James T.L. Houghten R.A. Cohen F.E. Prusiner S.B. Burton D.R. J. Mol. Biol. 1997; 273: 614-622Crossref PubMed Scopus (319) Google Scholar, 15Paramithiotis E. Pinard M. Lawton T. LaBoissiere S. Leathers V.L. Zou W.Q. Estey L.A. Lamontagne J. Lehto M.T. Kondejewski L.H. Francoeur G.P. Papadopoulos M. Haghighat A. Spatz S.J. Head M. Will R. Ironside J. O'Rouke K. Tonelli Q. Ledebur H.C. Chakrabartty A. Cashman N.R. Nat. Med. 2003; 9: 893-899Crossref PubMed Scopus (240) Google Scholar, 27Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar, 28Leclerc E. Peretz D. Ball H. Sakurai H. Legname G. Serban A. Prusiner S.B. Burton D.R. Williamson R.A. EMBO J. 2001; 20: 1547-1554Crossref PubMed Scopus (57) Google Scholar, 29Khalili-Shirazi A. Summers L. Linehan J. Mallinson G. Anstee D. Hawke S. Jackson G.S. Collinge J. J. Gen. Virol. 2005; 86: 2635-2644Crossref PubMed Scopus (56) Google Scholar). Using an array of specific antibodies recognizing different PrP epitopes and PrP conformations, we have developed a novel immunoconformational assay referred to as the dual color assay. In contrast to previously established techniques, the newly developed assay probes the conformation within a single fibril or particle of aggregated PrP. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) the regions of PrP that are surface-exposed or buried in aggregated forms of the protein, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of fibrils measured across either the whole fibrillar population or within individual fibrils. Protein Expression, Purification, and Conversion into Amyloid Fibril—Full-length mouse recombinant PrP encompassing residues 23–230 (rPrP) was expressed and purified as described earlier (30Bocharova O.V. Breydo L. Parfenov A.S. Salnikov V.V. Baskakov I.V. J. Mol. Biol. 2005; 346: 645-659Crossref PubMed Scopus (230) Google Scholar). The purified rPrP was confirmed by SDS-PAGE, analytical size exclusion chromatography, and electrospray mass spectrometry to be a single monomeric species with an intact disulfide bond. In vitro conversion of rPrP to amyloid fibrils was carried out under standard conditions (in 1 m GndHCl, 3 m urea in 20 mm sodium acetate buffer, pH 5.0, at 37 °C) as previously described (30Bocharova O.V. Breydo L. Parfenov A.S. Salnikov V.V. Baskakov I.V. J. Mol. Biol. 2005; 346: 645-659Crossref PubMed Scopus (230) Google Scholar). At the end point of conversion (typically 24 h of incubation) the reaction was stopped, and rPrP fibrils were dialyzed overnight in 10 mm sodium acetate buffer, pH 5.0, and stored at +4 °C. Double Color Assay—Amiloid fibrils and β-oligomeric forms of rPrP (0.5 μg/ml) were incubated for 1 h in Permanox 8-well Lab-Teks chamber slide system (200 μl/well) in 10 mm sodium acetate buffer, pH 5.0, at room temperature. Formaldehyde was then added to a final concentration of 4% and incubated for 15 min, and the samples were washed with Tris-buffered saline (TBS) containing 50 mm glycine, pH 7.5, to reduce the autofluorescence background of the aldehydes. In GdnHCl-induced denaturation experiments, samples were treated with 4 or 6 m GdnHCl, as indicated, in 50 mm Tris-HCl, pH 7.5, for 1 h and then washed with TBS prior to assaying. Pairs of anti-PrP antibodies were applied in two consecutive steps. First, samples were incubated for 1 h at room temperature with one of the following anti-PrP human Abs: D13 (1:6000), D18 (1:6000), 89–112 (1:6000), 136–158 (1:6000), or R1 (1:400). This was followed by incubation for 1 h at room temperature with a secondary Ab, goat anti-human IgG (1:700), labeled with Alexa-488 (Invitrogen/Molecular Probes). Second, the samples were incubated for 1 h at room temperature with a reference Ab, mouse IgG AG4 (1:1000), that recognizes epitope 37–50 in PrP, followed by incubation with secondary Ab, goat anti-mouse IgG (1:700), labeled with Alexa-546 (Invitrogen/Molecular Probes). In another combination, anti-PrP mouse IgG AH6 (1:400) was applied first and then the secondary Ab, goat anti-mouse IgG (1:700), labeled with Alexa-488, followed by incubation with anti-PrP human Fab P (1:1000) that was used as a reference antibody and then with a secondary Ab to Fab, goat anti-human IgG (1:700)-labeled Alexa-546. Staining with the primary antibodies was in TBS containing 0.25% Triton X-100, 5% normal horse serum, and 1% bovine serum albumin, and staining with the secondary antibodies was done in TBS containing 0.25% Triton X-100 and 2% bovine serum albumin. All antibodies were centrifuged for 10 min at 10,000 rpm prior to staining; 0.25% Triton X-100 was present throughout the whole staining procedure. Slides were mounted with antifade fluorescence mounting medium (DAKO, Denmark). Immunofluorescence Imaging and Data Analysis—Fluorescence microscopy was carried out on an inverted microscope (Nikon Eclipse TE2000-U) with an illumination system X-Cite 120 (EXFO Photonics Solutions Inc.) connected through fiber optics using a 1.3 aperture Plan Fluor ×100 numerical aperture and ×60 objectives. Digital images were acquired using a cooled 12-bit CoolSnap HQ CCD camera (Photometrics). The exposure time for AG4 was 700 ms; for D13, D18, 89–112, and 136–158 it was 1400 ms; for P it was 800 ms; and for R1 and AH6 it was 1600 ms. The excitation irradiance was reduced twice when images were collected with a ×60 objective. Images collected from the two channels were processed with WCIF ImageJ software (National Institutes of Health). Manders' Coefficients and Colocalization Threshold plugins were used to obtain two-dimensional fluorescence intensity scattering plots (2D-FIS). The colocalization threshold plugin was also used to calculate the values of linear regression slopes (LRS). S.D. of LRS values were calculated from the analysis of 3–5 images from the same experiment. Analysis of different subpopulations of fibrils observed after staining with Abs 89–112 or 136–158 was performed using the Colocalization Finder plugin, which allowed us to highlight and select the fibrils with a specified ratio of intensities measured in the “red” and “green” channels. Intensity profiles of single fibrils were built with the RGB profiler plugin. Design of the Dual Color Immunoconformational Assay—Our assay consisted of double staining using different pairings of PrP-specific antibodies, where the reference Ab was specific to the epitope that is solvent-accessible, but the second Ab was specific to the epitope for which we wanted to assess the conformation (Fig. 2a). Because in PrPSc and in the fibrillar form of rPrP, the N-terminal region encompassing residues 23 to ∼90 is proteinase K-sensitive and solvent-accessible (31Bocharova O.V. Breydo L. Salnikov V.V. Gill A.C. Baskakov I.V. Protein Sci. 2005; 14: 1222-1232Crossref PubMed Scopus (83) Google Scholar), we used AG4 antibody specific to the epitope 37–59 as a reference Ab (Fig. 1). AG4 was used in pairs with one of the following Abs: D13 (epitope 95–105), D18 (epitope 132–156), R1 (epitope 224–230), 89–112, or 136–158 (Fig. 1, Table 1). IgGs 89–112 and 136–158 were previously shown to bind specifically to PrPSc but not to PrPC (32Moroncini G. Kanu N. Solforosi L. Abalos G. Telling G.C. Head M. Ironside J. Brockes J.P. Burton D.R. Williamson R.A. Proc. Acad. Natl. Sci. U. S. A. 2004; 101: 10404-10409Crossref PubMed Scopus (100) Google Scholar). These Abs seem to recognize nonlinear epitopes, the locations of which are currently unknown (Fig. 1) (32Moroncini G. Kanu N. Solforosi L. Abalos G. Telling G.C. Head M. Ironside J. Brockes J.P. Burton D.R. Williamson R.A. Proc. Acad. Natl. Sci. U. S. A. 2004; 101: 10404-10409Crossref PubMed Scopus (100) Google Scholar). The secondary Ab to AG4 was labeled with Alexa-546 (red), and the secondary Ab to the remaining PrP specific Abs was labeled with Alexa-488 (green) (Fig. 2, a and b). When fluorescence microscopy was used for imaging, the double staining and subsequent merge of images collected in the red and green channels provided information regarding the solvent accessibility of the epitope of interest within individual fibrils (Fig. 2, b and c). A predominantly red color in the merged images indicated that the epitope of interest was largely buried; predominantly green color meant that the epitope of interest was solvent-accessible; whereas different tints of yellow and orange reflect slight differences in the accessibility of epitopes to the solvent (Fig. 2, b and c, bottom panels).FIGURE 1A schematic representation of full-length rPrP 23–230. The known binding epitopes of Fabs D13, P, D18, R1, and Abs AH6 and AG4 are highlighted in light gray. When converted into fibrillar form, the PK-resistant cores consist of residues 138–230, 152–230, and 162–230, highlighted as dark gray boxes (31Bocharova O.V. Breydo L. Salnikov V.V. Gill A.C. Baskakov I.V. Protein Sci. 2005; 14: 1222-1232Crossref PubMed Scopus (83) Google Scholar). Fabs 89–112 and 136–158 were generated by grafting mouse PrP sequences that correspond to amino acid residues 88–111 and 135–157, respectively, to recipient Ab (32Moroncini G. Kanu N. Solforosi L. Abalos G. Telling G.C. Head M. Ironside J. Brockes J.P. Burton D.R. Williamson R.A. Proc. Acad. Natl. Sci. U. S. A. 2004; 101: 10404-10409Crossref PubMed Scopus (100) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1The reactivity of the antibody to the amyloid fibrils as probed in the dual color assayAbEpitopeReference AbImmunoreactivityNo GdnHCl4 m GdnHClD1395-105AG4++++++D18132-156AG4-++AH6159-174P--/+R1224-230AG4--/+89-112aThe reactivity of IgG 89-112 is presented for the subpopulation of 89-112-positive fibrils.PrPSc-specificAG4+++-136-158bThe reactivity of IgG 136-158 is presented for the subpopulation of 136-158-positive fibrils.PrPSc-specificAG4+++++a The reactivity of IgG 89-112 is presented for the subpopulation of 89-112-positive fibrils.b The reactivity of IgG 136-158 is presented for the subpopulation of 136-158-positive fibrils. Open table in a new tab In the Fibrillar Form, the Epitope 95–105 Was Solvent-accessible, whereas the Epitope 132–156 Was Buried—Double staining of amyloid fibrils with AG/D13 revealed that under native conditions the epitope 95–105 was solvent-accessible (Fig. 3a). To analyze the fluorescence intensities in more detail, the microscopy images were transformed into 2D-FIS plots, where red fluorescence intensities are plotted on the horizontal axis, and the green intensities are plotted on the vertical axis (see insets to Fig. 3). As evident from the 2D-FIS plots, the amyloid fibrils consisted of a single population, which was relatively homogeneous with respect to the accessibility of the D13 epitope. From the 2D-FIS plot, the LRS can be calculated, a parameter that we used to measure the intrinsic accessibility of a particular epitope to a solvent. The LRS value of 1.307 ± 0.073 indicates that the D13 epitope is solvent-exposed under native conditions (Fig. 3a). The reactivity of D18 to PrP amyloid fibrils was substantially lower than that of D13, as reflected by the predominantly red color in microscopy images and by the low value of the LRS (0.349 ± 0.037) calculated from the 2D-FIS plot (Fig. 3b). Taken together, these data show that the epitope 132–156 was predominantly buried under the native conditions, whereas the epitope 95–105 was solvent-accessible (Table 1). To determine whether partial denaturation alters the accessibility of 95–105 and 132–156 epitopes, the fibrils were exposed to 4 m GdnHCl for 1 h prior to the dual color assay. We previously showed that the C½ value for the GdnHCl-induced denaturation of amyloid fibrils produced in vitro was 4.2 m (31Bocharova O.V. Breydo L. Salnikov V.V. Gill A.C. Baskakov I.V. Protein Sci. 2005; 14: 1222-1232Crossref PubMed Scopus (83) Google Scholar); therefore, at 4 m GdnHCl, the amyloid fibrils were expected to preserve most of the cross-β-sheet structure; however, some of the fibrillar regions could undergo local unfolding. Upon partial denaturation, the color distribution in samples stained with D18 shifted toward the “green” sector, as was evident from significant shift of the LRS value from 0.349 ± 0.037 to 0.845 ± 0.139 and from the broadening of the fluorescence intensity distribution in the 2D-FIS plot (Fig. 3d). These changes indicated that the D18 epitope that was cryptic under the native conditions became partially exposed to the solvent upon partial denaturation. In contrast to the D18 epitope, the accessibility of the noncryptic D13 epitope remained the same (LRS value unchanged) regardless of whether amyloid fibrils were preincubated with GdnHCl (Fig. 3, compare a and c, Table 1). Highly Denaturing Conditions Were Required to Expose Epitopes 159–174 and 224–230—Our former studies revealed that in the fibrillar form, the PK-resistant cores are constructed from the C-terminal regions that encompass residues 152–230 and 162–230 (31Bocharova O.V. Breydo L. Salnikov V.V. Gill A.C. Baskakov I.V. Protein Sci. 2005; 14: 1222-1232Crossref PubMed Scopus (83) Google Scholar). These C-terminal regions are believed to compose the most thermodynamically stable cross-β-sheet structures of fibrils. To test the accessibility of epitopes within the C-terminal region, we used IgG AH6 and Fab R1, which bind to the residues 159–174 and 224–230, respectively (Fig. 1). Here, to avoid unwanted cross-reactivity with the secondary Ab to IgG, IgG AG4 (red channel) was used as a reference in a pair with Fab R1 (green channel), and Fab P (epitope 95–105, red channel) was paired with IgG AH6 (green channel) (Table 1). When stained with either R1 or AH6, the amyloid fibrils showed no detectable immunoreactivity in the green channels in the absence of GdnHCl, suggesting that both epitopes were completely buried in the fibrillar interior (Fig. 4, a and b, left panels). After incubation with 4 m GdnHCl, a subfraction of fibrils displayed a dotted pattern of fluorescence in the green channels when stained with either R1 or AH6 (Fig. 4, a and b, middle panels). Such dotted patterns could be due to the local exposure of epitopes to R1 or AH6 occurring at the fibrillar edges, at the sites of occasional fibrillar bending or fragmentation, or at the junction of several fibrils. The dotted pattern observed with R1 or AH6 was in sharp contrast to the relatively smooth and uniform pattern of fluorescence observed in fibrils that were pretreated with 4 m GdnHCl and stained with D18 (Fig. 4c). After exposure to a more severe denaturing environment (6 m GdnHCl), the number of spots detectable in green channels and the fluorescence intensity of the spots increased in fibrils stained with either R1 or AH6 (Fig. 4, a and b, right panels, as indicated by the green arrows). These reactive spots were often seen at the fibrillar edges or at sites of fibrillar junctions or overlaps. The appearance of large amorphous spots was indicative of extensive denaturation and loss of fibrillar shape. Notable differences in patterns of staining observed between D18 and either R1 or AH6 suggest strikingly different roles that the regions 132–156 and 159–230 play in fibrillar structure. The epitope to D18 became solvent-accessible under conditions where fibrils still maintained fibrillar shape, whereas full denaturation and loss of fibrillar shape seem to be required for the R1 and AH6 epitopes to be solvent-accessible. PrPSc-specific Fabs Distinguished Two Subpopulations in Fibrils—To probe the extent to which the amyloid fibrils produced in vitro are similar to PrPSc, we used IgGs 89–112 and 136–158. Both IgGs 89–112 and 136–158 were previously shown to bind specifically to PrPSc but not to PrPC (32Moroncini G. Kanu N. Solforosi L. Abalos G. Telling G.C. Head M. Ironside J. Brockes J.P. Burton D.R. Williamson R.A. Proc. Acad. Natl. Sci. U. S. A. 2004; 101: 10404-10409Crossref PubMed Scopus (100) Google Scholar). These motif-grafted Abs were generated by replacing the complementarity-determining region 3 in heavy chains of recipient Ab with mouse PrP sequences that correspond to amino acid residues 88–111 and 135–157, respectively (32Moroncini G. Kanu N. Solforosi L. A" @default.
• W2018542940 created "2016-06-24" @default.
• W2018542940 creator A5007754320 @default.
• W2018542940 creator A5020839983 @default.
• W2018542940 creator A5025508579 @default.
• W2018542940 creator A5032924150 @default.
• W2018542940 creator A5034172810 @default.
• W2018542940 creator A5042499250 @default.
• W2018542940 creator A5083921697 @default.
• W2018542940 date "2006-06-01" @default.
• W2018542940 modified "2023-10-06" @default.
• W2018542940 title "Probing the Conformation of the Prion Protein within a Single Amyloid Fibril Using a Novel Immunoconformational Assay" @default.
• W2018542940 cites W1619710608 @default.
• W2018542940 cites W1938969372 @default.
• W2018542940 cites W1966382960 @default.
• W2018542940 cites W1978525961 @default.
• W2018542940 cites W1983292653 @default.
• W2018542940 cites W1984542187 @default.
• W2018542940 cites W1985315884 @default.
• W2018542940 cites W1986735189 @default.
• W2018542940 cites W1996513738 @default.
• W2018542940 cites W2004098528 @default.
• W2018542940 cites W2022614447 @default.
• W2018542940 cites W2023529808 @default.
• W2018542940 cites W2026460791 @default.
• W2018542940 cites W2033638305 @default.
• W2018542940 cites W2036014001 @default.
• W2018542940 cites W2057293794 @default.
• W2018542940 cites W2058032460 @default.
• W2018542940 cites W2060983898 @default.
• W2018542940 cites W2062743922 @default.
• W2018542940 cites W2063690703 @default.
• W2018542940 cites W2072303997 @default.
• W2018542940 cites W2077631732 @default.
• W2018542940 cites W2092556927 @default.
• W2018542940 cites W2095446645 @default.
• W2018542940 cites W2096299698 @default.
• W2018542940 cites W2122294662 @default.
• W2018542940 cites W2127178575 @default.
• W2018542940 cites W2133145715 @default.
• W2018542940 cites W2142524352 @default.
• W2018542940 cites W2144825332 @default.
• W2018542940 cites W2149403782 @default.
• W2018542940 cites W2154787186 @default.
• W2018542940 cites W2161471588 @default.
• W2018542940 cites W2162237536 @default.
• W2018542940 cites W2166409332 @default.
• W2018542940 cites W2167517570 @default.
• W2018542940 cites W2167564565 @default.
• W2018542940 cites W218529424 @default.
• W2018542940 cites W2327641970 @default.
• W2018542940 doi "https://doi.org/10.1074/jbc.m601349200" @default.
• W2018542940 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16569635" @default.
• W2018542940 hasPublicationYear "2006" @default.
• W2018542940 type Work @default.
• W2018542940 sameAs 2018542940 @default.
• W2018542940 citedByCount "63" @default.
• W2018542940 countsByYear W20185429402012 @default.
• W2018542940 countsByYear W20185429402013 @default.
• W2018542940 countsByYear W20185429402014 @default.
• W2018542940 countsByYear W20185429402015 @default.
• W2018542940 countsByYear W20185429402017 @default.
• W2018542940 countsByYear W20185429402019 @default.
• W2018542940 countsByYear W20185429402020 @default.
• W2018542940 countsByYear W20185429402022 @default.
• W2018542940 crossrefType "journal-article" @default.
• W2018542940 hasAuthorship W2018542940A5007754320 @default.
• W2018542940 hasAuthorship W2018542940A5020839983 @default.
• W2018542940 hasAuthorship W2018542940A5025508579 @default.
• W2018542940 hasAuthorship W2018542940A5032924150 @default.
• W2018542940 hasAuthorship W2018542940A5034172810 @default.
• W2018542940 hasAuthorship W2018542940A5042499250 @default.
• W2018542940 hasAuthorship W2018542940A5083921697 @default.
• W2018542940 hasBestOaLocation W20185429401 @default.
• W2018542940 hasConcept C12554922 @default.
• W2018542940 hasConcept C142724271 @default.
• W2018542940 hasConcept C179104552 @default.
• W2018542940 hasConcept C185592680 @default.
• W2018542940 hasConcept C27523624 @default.
• W2018542940 hasConcept C2777633098 @default.
• W2018542940 hasConcept C2779134260 @default.
• W2018542940 hasConcept C2909371084 @default.
• W2018542940 hasConcept C2994168385 @default.
• W2018542940 hasConcept C3019447875 @default.
• W2018542940 hasConcept C3019804061 @default.
• W2018542940 hasConcept C55493867 @default.
• W2018542940 hasConcept C71924100 @default.
• W2018542940 hasConcept C86803240 @default.
• W2018542940 hasConceptScore W2018542940C12554922 @default.
• W2018542940 hasConceptScore W2018542940C142724271 @default.
• W2018542940 hasConceptScore W2018542940C179104552 @default.
• W2018542940 hasConceptScore W2018542940C185592680 @default.
• W2018542940 hasConceptScore W2018542940C27523624 @default.
• W2018542940 hasConceptScore W2018542940C2777633098 @default.
• W2018542940 hasConceptScore W2018542940C2779134260 @default.
• W2018542940 hasConceptScore W2018542940C2909371084 @default.
• W2018542940 hasConceptScore W2018542940C2994168385 @default.
• W2018542940 hasConceptScore W2018542940C3019447875 @default.

• next