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Studies on methods for specific detection of abnormal isoform of prion protein and its application

鈴木, 章夫 北海道大学

2021.12.24

概要

プリオン病の病原体であるプリオンは,異常型プリオンタンパク質 (PrPSc) から構成される。PrPSc は,宿主由来正常型プリオンタンパク質 (PrPC) から構造転換を含む翻訳後修飾により産生される。PrPC と PrPSc の結合,および,その後の α-ヘリックスに富む PrPC からβ 構造に富むPrPSc への構造転換は,プリオン増殖において鍵となる事象であると考えられている。プリオン感染後,PrPSc の産生が始まり,長い潜伏期間の間に中枢神経系に徐々に蓄積する。最終的に,PrPSc の産生自体,または PrPSc の形成や蓄積に対する応答による神経組織環境の変化により神経細胞死が引き起こされる。PrPSc はプリオンの主要構成要素であり,PrPSc の産生過程はプリオンの増殖とみなされているため,PrPSc の特異的な検出は,プリオン増殖やプリオン病の神経病態機序の解析に重要である。さらに,PrPSc 特異的検出は,プリオン病の診断やサーベイランスにも重要である。そこで本学位論文では,PrPSc 特異的染色に非常に有用であるモノクローナル抗体 (mAb) 132 による PrPSc 特異的検出のメカニズムと,組換えシカ PrP (rCerPrP) を基質として用いた Real-Time Quaking-Induced Conversion (RT-QuIC) によるPrPSc 検出の有用性について研究した。

第一章では,mAb 132 による PrPSc 特異的検出のメカニズムを解明するため,一価および二価の mAb 132 の組換えマウスPrP (rMoPrP) に対する反応性を ELISA,表面プラズモン共鳴法 (SPR) により解析した。ELISA 法では,mAb 132 の一価での結合は二価型での結合に比べ有意に弱いことから,二価での結合が mAb 132 に高い結合安定性を与えていることが示唆された。また,他の抗 PrP 抗体に比べ mAb 132 の二価結合は,抗原濃度の減少により容易に影響を受けることが明らかとなった。SPR 法では,mAb 132の反応速度論は ELISA による結果と一致しており,一価結合の解離速度定数は二価結合より 260 倍大きかった。これは一価結合は二価結合より安定性が低い事を示唆している。さらに,抗原密度が低く抗体が二価結合できない場合,rMoPrP に結合したmAb 132 の量は減少した。一方,2つの PrPC が二価結合が可能な距離内に存在する場合,mAb 132 は PrPC に結合した。これらの結果は,単量体 PrPC への弱い一価結合が PrPC のシグナルをバックグラウンドレベルに減少させるのに対し,mAb 132 は PrPSc のエピトープ露出後に二価結合により PrPSc に安定して結合する事を示している。

第二章では,rCerPrP を含む様々な組換え PrP (rPrP) を用いて,脳乳剤濃度が高い場合でも低レベルの慢性消耗病 (CWD),牛海綿状脳症 (BSE) プリオンを検出可能なRT-QuIC の反応条件について検討した。試したrPrP のうち,rCerPrP のみが,CWD,非定型BSE プリオンに対する反応性が高濃度の非感染脳乳剤による影響を受けにくいという特徴的な反応性を示した。この rCerPrP 特有の反応性は,N 末端領域アミノ酸 25−93を除去することにより消失した。また,MoPrP のアミノ酸 23−149 をCerPrP の対応する領域と置換することで,RT-QuIC 反応での CerPrP 特有の反応性が部分的に回復した。さらに, MoPrP の C 末端領域アミノ酸 219−231 を CerPrP の対応する領域と置換すると rCerPrP特有の反応性がrMoPrP に付与されたことから,N 末端,C 末端領域の両方が rCerPrP 特有の反応性に関与することが示唆された。加えて,CerPrP のアミノ酸 25−153,MoPrP のアミノ酸150−218,およびCerPrP のアミノ酸223−233 から構成されるrCerN−Mo−CerCPrP は,この N 末端,C 末端領域の相加効果を示した。これらの結果は,CWD および非定型BSEを検出する際の rCerPrP の作用機序を示すとともに,RT-QuIC の更なる改善に役立つと思われる。

第三章では,日本におけるCWD の状態を明らかにするために,全長rCerPrP を使用した RT-QuIC による CWD モニタリングを行った。北海道,本州で捕獲または狩猟された合計 690 個のニホンシカ,ジャワマメジカの延髄閂部サンプルを RT-QuIC により検査した。CWD 陽性例は認められなかったことから,CWD は日本で発生していない可能性が高いと考えられる。さらに,ニホンシカの PrP 遺伝子の塩基配列の解析により,日本の広い地域に生息するニホンシカが CWD 感受性の個体群により構成されていることが示唆された。従って,より広い地域のシカについての継続的なモニタリングは,CWD の発生時における早期の検出及び対応やシカ由来製品の安全性を確保するために必要と思われる。

本学位論文では,抗 PrP 抗体 mAb 132 による PrPSc 特異的染色のメカニズムを解明した。これらの知見は,プリオンの増殖およびプリオン病の神経病態機序を明らかにするための mAb 132 の使用に対する論理的根拠を提供しうる。また,RT-QuIC 反応において CWD と BSE を検出する際の rCerPrP の有用性について示した。これらの結果は, RT-QuIC の実用性を示すとともに,RT-QuIC の更なる改善にも役立つと思われる。PrPSc特異的検出及びその応用についての知見は,プリオン病の神経病態学の解明や,プリオン病の診断,サーベイランスの更なる改善に貢献するものである。

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