1998 Fiscal Year Final Research Report Summary
In Vitro studies on the formation of prion amyloid
| Project/Area Number |
08456145
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Basic veterinary science/Basic zootechnical science
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| Research Institution | Obihiro University of Agriculture and Veterinary Medicine |
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Principal Investigator |
SHINAGAWA Morikazu Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, Professor, 畜産学部, 教授 (00001537)
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| Co-Investigator(Kenkyū-buntansha) |
KUWAYAMA Hideto Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, A, 畜産学部, 助教授 (40125399)
ISHIGURO Naotaka Obihiro University of Agriculture and Veterinary Medicine・Veterinary Medicine, A, 畜産学部, 助教授 (00109521)
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| Project Period (FY) |
1996 – 1998
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| Keywords | prion amvloid / in vitro structural conversion / recombinant PrP^c / proteinase K resistance / CD spectrum |
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| Research Abstract |
As a final goal of this study is in vitro formation of infectious prion amyloid using mouse PrP^C, in vitro structural conversion of PrP^C using a small amount of mouse prion was carried out. To eliminate the effects of unknown mouse protein contaminating in PrP^C, mouse recombinant PrP^C which possessed a histidine tag at N-terminus of mature PrP^C was used. The recombinant PrP^C converted to a proteinase K (PK) resistant form in the presence of one-thousandth amounts of mouse scrapie prion at pH 5.2 and room temperature for one day and after 14 days the PK-resistance increased more. A decrease of alpha-helix contents estimated by CD spectrum and an increase of beta-sheet contents estimated by Congo red binding were observed in the PK-resistant recombinant PrP^C, while the recombinant PrP^C incubated for 14 days without mouse prion showed a slight increase of PK-resistance and no change in alpha-helix and beta-sheet contents. These facts indicate that structural conversion occurred in the recombinant PrP^C in the presence of mouse prion. The conversion was also induced by adding one-hundredth amounts of the PK-resistant recombinant PrP^C, but was inhibited by adding 40 mM of a synthetic peptide corresponding to mouse prion codons 113-141. The molecular weight of the PK-resistant recombinant PrP^C did not change after PK-treatment but that of prion polypeptide decreases after PK-treatment in polyacrylamide gel electrophoresis. This indicates that the structure of the PK-resistant recombinant PrP^C differed from that of prion. In addition to PrP^C and prion, some unknown factors may be required to form prion amyloid. Other studies in relation to this theme also have been done.
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[Publications] Laplanche, J-L., Hunter, N., Shinagawa, M., Williams, E.In Prion Biology and Diseases(Prusiner, S.B.ed): Scrapie, Chronic Wasting Disease, and Transmissible Mink Encephalopathy.Cold Spring Harbor Laboratory Press(in press),
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