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Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins
The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). Diverse mammalian species possess the cofactors required for in vitro replication of PrP(Sc) by protein-misfolding cyclic amplification (PMCA),...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867355/ https://www.ncbi.nlm.nih.gov/pubmed/24367521 http://dx.doi.org/10.1371/journal.pone.0082538 |
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author | Imamura, Morikazu Kato, Nobuko Okada, Hiroyuki Yoshioka, Miyako Iwamaru, Yoshifumi Shimizu, Yoshihisa Mohri, Shirou Yokoyama, Takashi Murayama, Yuichi |
author_facet | Imamura, Morikazu Kato, Nobuko Okada, Hiroyuki Yoshioka, Miyako Iwamaru, Yoshifumi Shimizu, Yoshihisa Mohri, Shirou Yokoyama, Takashi Murayama, Yuichi |
author_sort | Imamura, Morikazu |
collection | PubMed |
description | The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). Diverse mammalian species possess the cofactors required for in vitro replication of PrP(Sc) by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrP(Sc) seeds and Bac-PrP(Sc) generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrP(Sc) were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrP(Sc) of some prion strains. |
format | Online Article Text |
id | pubmed-3867355 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-38673552013-12-23 Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins Imamura, Morikazu Kato, Nobuko Okada, Hiroyuki Yoshioka, Miyako Iwamaru, Yoshifumi Shimizu, Yoshihisa Mohri, Shirou Yokoyama, Takashi Murayama, Yuichi PLoS One Research Article The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). Diverse mammalian species possess the cofactors required for in vitro replication of PrP(Sc) by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrP(Sc) seeds and Bac-PrP(Sc) generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrP(Sc) were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrP(Sc) of some prion strains. Public Library of Science 2013-12-18 /pmc/articles/PMC3867355/ /pubmed/24367521 http://dx.doi.org/10.1371/journal.pone.0082538 Text en © 2013 Imamura et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Imamura, Morikazu Kato, Nobuko Okada, Hiroyuki Yoshioka, Miyako Iwamaru, Yoshifumi Shimizu, Yoshihisa Mohri, Shirou Yokoyama, Takashi Murayama, Yuichi Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title | Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title_full | Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title_fullStr | Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title_full_unstemmed | Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title_short | Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins |
title_sort | insect cell-derived cofactors become fully functional after proteinase k and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and bse prion proteins |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867355/ https://www.ncbi.nlm.nih.gov/pubmed/24367521 http://dx.doi.org/10.1371/journal.pone.0082538 |
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