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Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein*

  • Toshiya Kubota
    Affiliations
    Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan
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  • Yuta Hamazoe
    Affiliations
    Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan
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  • Shuhei Hashiguchi
    Affiliations
    Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan
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  • Daisuke Ishibashi
    Affiliations
    Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Science, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
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  • Kazuyuki Akasaka
    Affiliations
    Department of Biotechnological Science, School of Biology-oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa-shi, Wakayama 649-6493, Japan
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  • Noriyuki Nishida
    Affiliations
    Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Science, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
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  • Shigeru Katamine
    Affiliations
    Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Science, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
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  • Suehiro Sakaguchi
    Affiliations
    Division of Molecular Neurobiology, The Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan
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  • Ryota Kuroki
    Affiliations
    Neutron Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirane Shirakata, Tokai, Naka-gun, Ibaraki 319-1195, Japan
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  • Toshihiro Nakashima
    Affiliations
    Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto 869-1298, Japan
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  • Kazuhisa Sugimura
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 1-21-40 Korimoto, Kagoshima 890-0065, Japan. Tel.: 81-99-285-8345; Fax: 81-99-258-4706;
    Affiliations
    Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Japan
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  • Author Footnotes
    * This work was supported by Grants-in-aid for Scientific Research C 16613008 and for Young Scientists B 22790435 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S. H.), Health and Labour Sciences Research Grants on Food Safety (Shokuhin-016) (to S. H.) and Psychiatric and Neurological Diseases and Mental Health from the Ministry of Health, Labour and Welfare (to S. H. and K. S.), a grant-in-aid from the Bovine Spongiform Encephalopathy Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan (to K. S.), and Super Special Consortia for supporting the development of cutting-edge medical care from 2008 to 2012 (to K. S.).
    This article contains supplemental Figs. S1–S7.
Open AccessPublished:February 22, 2012DOI:https://doi.org/10.1074/jbc.M111.318352
      We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935–1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrPSc with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

      Introduction

      Prion diseases are fatal and transmissible neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. It has been proposed that a misfolded form of prion protein is responsible for the infectivity of prion disease, and the pathogenesis of prion disease involves a conformational change of prion protein (PrP)
      The abbreviations used are: PrP
      prion protein
      PK
      proteinase K
      Vhls
      heavy-chain leader sequence
      Vh
      heavy-chain variable gene
      Vl
      light-chain variable gene
      Vlls
      light-chain leader sequence
      scFv
      single chain Fv(s)
      IGHC1
      human immunoglobulin heavy chain 1
      IGKC
      human immunoglobulin κ-chain
      ScN2a
      Scrapie-infected neuroblastoma
      rPrP
      recombinant full-length prion protein
      GdnHCl
      guanidine hydrochloride
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      SEC
      size exclusion chromatography.
      from PrPC to PrPSc (
      • Prusiner S.B.
      Prions.
      ). PrPC is a monomeric isoform, rich in α-helical structure, and sensitive to digestion by proteinase K (PK). In contrast, multimers are PrPSc characterized as PrPres by enhanced resistance toward PK digestion. Consequently, it is believed first that PrPSc might have a β-pleated sheet structure because all amyloids studied have been found to have this structure and second that this β-sheet-rich PrPSc binds specifically to PrPC, propagating its altered conformation via a templating mechanism, which is a prion activity. In other words, it was demonstrated that PrPSc, defined as PrP27–30, was generated and accumulated in prion-infected cells or brain. However, there is no direct evidence of whether prion or PrPSc has a β-sheet-rich structure, although there is suggestive evidence of FTIR using centrifugation-purified aggregates of prion-infected brain extracts (
      • Rogers M.
      • Yehiely F.
      • Scott M.
      • Prusiner S.B.
      Conversion of truncated and elongated prion proteins into the scrapie isoform in cultured cells.
      ,
      • Pan K.M.
      • Baldwin M.
      • Nguyen J.
      • Gasset M.
      • Serban A.
      • Groth D.
      • Mehlhorn I.
      • Huang Z.
      • Fletterick R.J.
      • Cohen F.E.
      Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins.
      ).
      Antibodies to these proteins are powerful tools to clarify these questions. However, 1) the purification procedure of PrPSc was based on the centrifugation precipitation of molecular aggregates using PEG or sodium phosphotungstic acid, which does not necessarily guarantee the specificity to a prion (
      • Rogers M.
      • Yehiely F.
      • Scott M.
      • Prusiner S.B.
      Conversion of truncated and elongated prion proteins into the scrapie isoform in cultured cells.
      ,
      • Prusiner S.B.
      • McKinley M.P.
      • Bowman K.A.
      • Bolton D.C.
      • Bendheim P.E.
      • Groth D.F.
      • Glenner G.G.
      Scrapie prions aggregate to form amyloid-like birefringent rods.
      ,
      • Turk E.
      • Teplow D.B.
      • Hood L.E.
      • Prusiner S.B.
      Purification and properties of the cellular and scrapie hamster prion proteins.
      ,
      • Biasini E.
      • Tapella L.
      • Mantovani S.
      • Stravalaci M.
      • Gobbi M.
      • Harris D.A.
      • Chiesa R.
      Immunopurification of pathological prion protein aggregates.
      ,
      • Wadsworth J.D.
      • Joiner S.
      • Hill A.F.
      • Campbell T.A.
      • Desbruslais M.
      • Luthert P.J.
      • Collinge J.
      Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.
      ). 2) The specificity of an anti-PrPSc antibody was defined by the existence of PrP27–30 resulting from PK-treated prion-infected brain or cell extracts that are immunoprecipitated with tested antibodies. These experiments showed that PrP27–30 was resolved from PK-resistant PrP but do not directly indicate that PrP27–30 is generated from β-sheet-rich PrP; i.e. they rather suggest a generation of β-sheet-rich PrP by experimental procedures including the use of PK, denaturing agents, or detergents (
      • Bocharova O.V.
      • Breydo L.
      • Parfenov A.S.
      • Salnikov V.V.
      • Baskakov I.V.
      In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc).
      ). 3) All antibodies established so far are either anti-PrPSc/PrPC or anti-PrPC antibodies (
      • Khalili-Shirazi A.
      • Kaisar M.
      • Mallinson G.
      • Jones S.
      • Bhelt D.
      • Fraser C.
      • Clarke A.R.
      • Hawke S.H.
      • Jackson G.S.
      • Collinge J.
      β-PrP form of human prion protein stimulates production of monoclonal antibodies to epitope 91–110 that recognise native PrPSc.
      ). More recently, mouse monoclonal IgG W261, which reacts exclusively with PrPSc but not PrPC, has been reported by cell fusion technology using spleen cells immunized with sodium phosphotungstic acid-precipitated PrPSc derived from prion-infected brain extracts (
      • Petsch B.
      • Müller-Schiffmann A.
      • Lehle A.
      • Zirdum E.
      • Prikulis I.
      • Kuhn F.
      • Raeber A.J.
      • Ironside J.W.
      • Korth C.
      • Stitz L.
      Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.
      ). This study did not address the question of whether PrPSc was the β-form PrP or not.
      In this study, using the conformation-defined recombinant PrPs and a human single chain Fv-displaying phage library, we have established two human IgG, PRB7 and PRB30, which are specific to the β-form but not the α-form of recombinant PrP of human, bovine, sheep, and mouse. Epitope mapping analysis showed that PRB7 IgG recognized residues 128–132 of the full-length prion protein.
      When prion-infected ScN2a cells were cultured in the presence of PRB7, apoptotic cells with numerous PRB7 binding signals including large aggregates were gradually generated during 4 days of culture. This finding is the first direct evidence of the generation and accumulation of β-sheet-rich prion protein in ScN2a cells. Interestingly, in these apoptotic cells, SAF32-staining granules were distinct from PRB7-binding aggregates, suggesting that SAF32-binding PrP does not have a PRB7-recognizing β-sheet structure, whereas PRB7-binding PrP may not have the N-terminal octarepeat region of PrP. After ScN2a cells were cultured in the presence of PRB7 or SAF32 for 3 days, PK-treated cell lysate was immunoblotted by 6D11 to examine the inhibitory effects of PRB7 IgG on the generation/accumulation of PrPSc. Surprisingly, PRB7 IgG had no influence, whereas SAF32 strongly inhibited the generation/accumulation of PrPres.
      Thus, this study reports the first establishment of a human IgG antibody recognizing β-form PrP but not α-form PrP and the use of this antibody to provide direct evidence of the de novo generation and conversion of β-sheet-rich PrP in prion-infected cells. PRB7 IgG can be a powerful tool to purify the β-form PrP generated de novo and demonstrate its biochemical basis and significance to elucidate structural evidence of prion infectivity and neurotoxicity.

      DISCUSSION

      A number of studies showed that prions generate and accumulate PrPSc, which shows resistance to PK degradation. It is postulated that PrPSc converts PrPC to PrPSc by its templating activity. PrPSc was purified as a multimer or aggregates. The protease-resistant core of PrPSc, designated PrP27–30, polymerizes into an amyloid. Many purified amyloids have been found to have a β-pleated sheet structure. From these findings, it is believed that prion or PrPSc may have a β-sheet-rich structure (
      • Pan K.M.
      • Baldwin M.
      • Nguyen J.
      • Gasset M.
      • Serban A.
      • Groth D.
      • Mehlhorn I.
      • Huang Z.
      • Fletterick R.J.
      • Cohen F.E.
      Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins.
      ,
      • Turk E.
      • Teplow D.B.
      • Hood L.E.
      • Prusiner S.B.
      Purification and properties of the cellular and scrapie hamster prion proteins.
      ).
      Is PrPSc a β-form PrP? Antibodies should be a powerful tool to solve this question. Despite the insufficient biochemical characterization of PrPSc, many attempts have been made to establish antibodies with fine specificity using mice immunized with partially purified PrPSc from prion-infected cells or brain extracts (
      • Biasini E.
      • Tapella L.
      • Mantovani S.
      • Stravalaci M.
      • Gobbi M.
      • Harris D.A.
      • Chiesa R.
      Immunopurification of pathological prion protein aggregates.
      ,
      • Petsch B.
      • Müller-Schiffmann A.
      • Lehle A.
      • Zirdum E.
      • Prikulis I.
      • Kuhn F.
      • Raeber A.J.
      • Ironside J.W.
      • Korth C.
      • Stitz L.
      Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.
      ,
      • Korth C.
      • Stierli B.
      • Streit P.
      • Moser M.
      • Schaller O.
      • Fischer R.
      • Schulz-Schaeffer W.
      • Kretzschmar H.
      • Raeber A.
      • Braun U.
      • Ehrensperger F.
      • Hornemann S.
      • Glockshuber R.
      • Riek R.
      • Billeter M.
      • Wüthrich K.
      • Oesch B.
      Prion (PrPSc)-specific epitope defined by a monoclonal antibody.
      ,
      • Ushiki-Kaku Y.
      • Endo R.
      • Iwamaru Y.
      • Shimizu Y.
      • Imamura M.
      • Masujin K.
      • Yamamoto T.
      • Hattori S.
      • Itohara S.
      • Irie S.
      • Yokoyama T.
      Tracing conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies.
      ,
      • Beringue V.
      • Vilette D.
      • Mallinson G.
      • Archer F.
      • Kaisar M.
      • Tayebi M.
      • Jackson G.S.
      • Clarke A.R.
      • Laude H.
      • Collinge J.
      • Hawke S.
      PrPSc binding antibodies are potent inhibitors of prion replication in cell lines.
      ,
      • Stanker L.H.
      • Serban A.V.
      • Cleveland E.
      • Hnasko R.
      • Lemus A.
      • Safar J.
      • DeArmond S.J.
      • Prusiner S.B.
      Conformation-dependent high-affinity monoclonal antibodies to prion proteins.
      ,
      • Skrlj N.
      • Vranac T.
      • Popović M.
      • Curin Šerbec V.
      • Dolinar M.
      Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment.
      ,
      • Kosmač M.
      • Koren S.
      • Giachin G.
      • Stoilova T.
      • Gennaro R.
      • Legname G.
      • Serbec V.Č.
      Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment.
      ,
      • Sasamori E.
      • Suzuki S.
      • Kato M.
      • Tagawa Y.
      • Hanyu Y.
      Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2.
      ,
      • Horiuchi M.
      • Karino A.
      • Furuoka H.
      • Ishiguro N.
      • Kimura K.
      • Shinagawa M.
      Generation of monoclonal antibody that distinguishes PrPSc from PrPC and neutralizes prion infectivity.
      ,
      • Saá P.
      • Castilla J.
      • Soto C.
      Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification.
      ,
      • Sandberg M.K.
      • Al-Doujaily H.
      • Sharps B.
      • Clarke A.R.
      • Collinge J.
      Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.
      ). However, most antibodies were specific to PrPC or cross-reactive to both PrPC and PrPSc but not monospecific to PrPSc, although more recently a PrPSc-specific murine IgG1 antibody, W261, has been established (
      • Petsch B.
      • Müller-Schiffmann A.
      • Lehle A.
      • Zirdum E.
      • Prikulis I.
      • Kuhn F.
      • Raeber A.J.
      • Ironside J.W.
      • Korth C.
      • Stitz L.
      Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.
      ). However, even in this situation, PrP conformation-specific antibodies have not been developed.
      In this study, using conformation-defined recombinant β-form PrP and an antibody-displaying phage library, we established a β-form PrP-specific human IgG1 antibody, PRB7 IgG. This antibody does not recognize generic oligomer or aggregate forms of unrelated proteins in accordance with the binding activity of PRB7 or PRB30 scFv. PRB7 IgG is the first human IgG antibody specifically binding to β-form PrP monomer and oligomers but not α-form PrP. It is suggested that PRB7 IgG recognizes the epitope 128–132 of β-form human PrP. PRB7 IgG is equally cross-reactive to the β-form of bovine, sheep, and mouse PrP.
      As PRB7 IgG does not recognize paraformaldehyde-fixed or denatured PrP molecules, we simply observed the ScN2a cells cultured with PRB7 IgG. We have demonstrated here that prion-infected ScN2a cells specifically internalized PRB7 IgG and accumulated the PrP granules bound with PRB7 IgG in the cytoplasm (Fig. 8). This is the first direct demonstration that β-form PrP was generated and accumulated in prion-infected cells in the physiological condition. Because neither ScN2a nor N2a58 cells were stained with normal IgG as assessed by confocal microscopy or flow cytometry, these cells were Fcγ receptor-negative. Nevertheless, ScN2a cells internalized PRB7 IgG, suggesting that complexes of β-form PrP targeted by PRB7 IgG localizing on the cell surface were endocytosed into the cytoplasm (
      • Veith N.M.
      • Plattner H.
      • Stuermer C.A.
      • Schulz-Schaeffer W.J.
      • Bürkle A.
      Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy.
      ). If this β-form PrP corresponds to PrPSc, this observation is in accordance with the finding that PrPSc was generated on a lipid raft on the cell surface (
      • Hnasko R.
      • Serban A.V.
      • Carlson G.
      • Prusiner S.B.
      • Stanker L.H.
      Generation of antisera to purified prions in lipid rafts.
      ). When ScN2a cells were cultured with PRB7 IgG, they gradually generated an increasing number of tiny PRB7-binding granules in the cytoplasm. Among them, about 0.4% of cells had enormous aggregates in the cytoplasm. Vertically severed images of these cells show traces of nuclei in cells that were weakly stained with Annexin V, indicating that they had undergone apoptosis (Fig. 10A). These findings suggested that an enormous aggregate resulted from the fusion of many tiny β-form granules primarily endocytosed from the cell surface but was not the result of β-form PrP propagation in the cytoplasm.
      SAF32 bound tiny granules dispersed in the cytoplasm that were distinct from large aggregates stained with PRB7 IgG (Fig. 10C). These results suggested first that PRB7-positive β-form PrPs lost or hid the N-terminal octarepeat region to which SAF32 bound and second that granules stained with SAF32 were not generated from β-form PrP.
      It was suggested that PrPC-PrPSc conversion, which is physiologically prevented by an energy barrier, may be a spontaneous stochastic event favored by mutations in the PRNP gene or acquired by infection with exogenous PrPSc. Accordingly, it is conceivable that GdnHCl treatment of cells additively induces the conformational conversion of PrP, leading to the formation of aggregates in experiments that immunohistologically attempted to show granules of PrPSc in the cytoplasm (
      • Bocharova O.V.
      • Breydo L.
      • Parfenov A.S.
      • Salnikov V.V.
      • Baskakov I.V.
      In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc).
      ). It is noted that when ScN2a cells were stained with SAF32 or 6D11 with a denaturing pretreatment by 6 m GdnHCl numerous tiny PrP granules became visible in the cytoplasm, and their images were quite distinct from those of PRB7-staining aggregates. Our results indicated that apoptotic cells showed two distinct types of PrP granules in the cytoplasm: one was SAF32-negative and PRB7 IgG-positive granules (N-terminal region-deleted; β-form PrP), and the other was PRB7 IgG-negative and SAF32-positive granules (full-length PrP lacking a β-sheet-rich conformation) (Fig. 10E). Related studies have reported that PK-sensitive PrPSc was contained in prion aggregates (
      • D'Castro L.
      • Wenborn A.
      • Gros N.
      • Joiner S.
      • Cronier S.
      • Collinge J.
      • Wadsworth J.D.
      Isolation of proteinase K-sensitive prions using pronase E and phosphotungstic acid.
      ) and more recently that there is heterogeneity in PrPSc conformers including small soluble PrP aggregates (
      • Biasini E.
      • Tapella L.
      • Mantovani S.
      • Stravalaci M.
      • Gobbi M.
      • Harris D.A.
      • Chiesa R.
      Immunopurification of pathological prion protein aggregates.
      ). Regarding the requirement of full-length PrP for prion activity, the answer is not consistent: i.e. Prusiner et al. (
      • Prusiner S.B.
      • Groth D.F.
      • Bolton D.C.
      • Kent S.B.
      • Hood L.E.
      Purification and structural studies of a major scrapie prion protein.
      ) reported that the purified PK-digested PrP27–30 was not infectious, whereas Anaya et al. (
      • Anaya Z.E.
      • Savistchenko J.
      • Massonneau V.
      • Lacroux C.
      • Andréoletti O.
      • Vilette D.
      Recovery of small infectious PrP(res) aggregates from prion-infected cultured cells.
      ) have recently reported that small infectious PrPres aggregates were recovered in the absence of strong in vitro denaturing treatments from prion-infected cultured cells. On the other hand, using an anti-aggregated PrP IgM antibody, 15B3, Biasini et al. (
      • Biasini E.
      • Tapella L.
      • Mantovani S.
      • Stravalaci M.
      • Gobbi M.
      • Harris D.A.
      • Chiesa R.
      Immunopurification of pathological prion protein aggregates.
      ,
      • Biasini E.
      • Seegulam M.E.
      • Patti B.N.
      • Solforosi L.
      • Medrano A.Z.
      • Christensen H.M.
      • Senatore A.
      • Chiesa R.
      • Williamson R.A.
      • Harris D.A.
      Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies.
      ) reported the successful purification of pathological full-length prion aggregates, and Cronier et al. (
      • Cronier S.
      • Gros N.
      • Tattum M.H.
      • Jackson G.S.
      • Clarke A.R.
      • Collinge J.
      • Wadsworth J.D.
      Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin.
      ) found that PK-sensitive PrPSc was involved in prion aggregates. Our findings in Fig. 10 suggest that PrP composed of tiny aggregates visualized with SAF32 or 6D11 after 6 m GdnHCl treatment of cells may be a full-length PrP with α-conformation.
      To examine the influence of PRB7 IgG on the templating activity of β-form PrP to accumulate PrPSc, ScN2a cells were cultured in the presence of PRB7 IgG or SAF32, and their PK-treated cell lysates were immunoblotted with 6D11 to evaluate the amount of PrPres. Surprisingly, PRB7 IgG had no effect, whereas SAF32 strongly inhibited the generation/accumulation of PrPSc. Similar results have been reported in which a PrPSc-specific antibody had no prion-clearing effect, whereas the PrPC-specific antibody showed marked clearing activity (
      • Petsch B.
      • Müller-Schiffmann A.
      • Lehle A.
      • Zirdum E.
      • Prikulis I.
      • Kuhn F.
      • Raeber A.J.
      • Ironside J.W.
      • Korth C.
      • Stitz L.
      Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.
      ). Because the mimotope of PRB7 is located at 128–132, if this β-pleated sheet forms an interface for prion aggregation PRB7 IgG may block the assembly of PrP aggregates because the binding IgG is much larger than the target molecule (PrP). We examined this activity using FF32 cells or N2aL1 cells and obtained the same results as for ScN2a cells (data not shown). In this respect, our result suggested that, in the prion conversion reaction, PRB7 IgG-recognizing β-form PrP did not play a role in driving the conversion of the PrP conformation and appears rather to be an end product that loses prion activity and accumulates in the cytoplasm. This end product may trigger cell cytotoxicity via the mitochondrial machinery (
      • Sandberg M.K.
      • Al-Doujaily H.
      • Sharps B.
      • Clarke A.R.
      • Collinge J.
      Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.
      ). This is in concert with the result that β-form recombinant PrP does not work as a template for prion amplification in protein misfolding cyclic amplification (
      • Saá P.
      • Castilla J.
      • Soto C.
      Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification.
      ,
      • Saborio G.P.
      • Permanne B.
      • Soto C.
      Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.
      ). It is conceivable that PRB7-negative, SAF32-positive granules as a full-length, non-β-form PrP might be responsible for prion propagation (Fig. 10, C and E). Our results may be in agreement with the finding that prion propagation and toxicity in vivo occur in two distinct mechanistic phases (
      • Sandberg M.K.
      • Al-Doujaily H.
      • Sharps B.
      • Clarke A.R.
      • Collinge J.
      Prion propagation and toxicity in vivo occur in two distinct mechanistic phases.
      ). Thus, we have not obtained evidence to determine whether PRB7-recognizing β-form PrP represents the so-called PrPSc that has both conversion activity and cell cytotoxicity. Further investigation is needed to determine whether β-form PrP affinity-purified with PRB7 shows prion activity such as the infectious and conversion/accumulation activity of the prion.
      Although a number of studies illuminated the accumulation of PrPres in prion-infected cells or brain extracts, there is little literature giving much attention to the phenomenon in which most prion-infected ScN2a cells vigorously proliferate to survive in culture (
      • Uryu M.
      • Karino A.
      • Kamihara Y.
      • Horiuchi M.
      Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones.
      ). The low frequency of PRB7 IgG-positive granules in ScN2a cells suggests the stochastic nature of the conversion of α-form to β-form PrP. The spongelike degeneration of prion-infected brain may reflect this feature.
      In conclusion, we report the establishment of PRB7 IgG, which is the first human antibody discriminating the β-form from the α-form of PrP in the physiological condition. Using this antibody, we have demonstrated direct evidence of the generation and accumulation of β-form PrP in prion-infected ScN2a cells. It can be used to purify β-form PrP molecules without the need for protease digestion and may be useful to demonstrate the biochemical basis of the relationship of β-form PrP to PrPSc and to elucidate the structure-based evidence for prion infectivity and neurotoxicity.

      Acknowledgments

      We thank Mayumi Yamamoto and Yuko Sato for the initial study on this theme.

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