JP5178009B2 - ボツリヌス神経毒素検出のための方法および複合体 - Google Patents
ボツリヌス神経毒素検出のための方法および複合体 Download PDFInfo
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Description
本発明は、国立衛生研究所による助成金番号第NIH GM56827号および第MH061876号に基づき、アメリカ合衆国政府の支援により成されたものである。したがって、アメリカ合衆国は本発明において特定の権利を有する。
(関連した出願の相互参照)
本出願は、2003年12月19日出願の米国仮出願第60/530,645号、および2004年6月15日出願の米国仮出願第60/579,254号の優先権を主張するものである。両仮出願の全てお内容は本文に引用されることによって組み込まれる。
(発明の背景)
ボツリヌス神経毒素(BoNT)はボツリヌス菌(Clostridium botulinum)により産生され、知られている中で最も強力な毒素である。この毒素は、食中毒の原因としてよく知られ、しばしば被害者たちに深刻な被害さらに死をももたらす。現在、構造上類似した7種のボツリヌス神経毒素または抗原型(BoNT/A型〜G型)があり、それぞれ重鎖(〜100KD)および軽鎖(〜50KD)から構成される。重鎖は、レセプターを介したエンドサイトーシスによる毒素が標的細胞へ進入することを媒介する。ひとたび取り込まれると、軽鎖は、エンドソーム小胞内腔から細胞質ゾルへと移行し、小胞を標的とした膜融合を仲介するタンパク質(「基質タンパク質」)を切断する亜鉛依存性プロテアーゼとして働く。SNAREタンパク質の切断が小胞と細胞膜の融合を阻害し、神経筋接合部での神経伝達物質の放出を阻止する。
(発明の概要)
一つの実施の形態において、本発明は、シナプトブレビン、シンタキシン、およびSNAP-25から成るグループより選択されるボツリヌス神経毒素の基質と、あるいはボツリヌス神経毒素により認識し切断されることができるそれらのフラグメント(「切断可能フラグメント」)であるリンカーペプチドと、10nM以上離れることなく配置する蛍光ドナー部分および蛍光受容部分とからなり、該蛍光ドナー部分の発光スペクトルが該蛍光受容部分の励起スペクトルとオーバーラップする、蛍光共鳴エネルギー転移(FRET)が可能な分子複合体を提供する。
(図の説明)
図1は、ボツリヌス神経毒素プロテアーゼ活性をモニターするためのCFP-YFPに基づいたバイオセンサーの概略図である。図1Aは、該バイオセンサー複合体の設計図である。CFPおよびYFPはそれぞれ、シナプトブレビン(アミノ酸33-94、上のパネル)あるいはSNAP-25(アミノ酸141-206、下のパネル)のフラグメントを介して結合している。これらのフラグメント上の各ボツリヌス神経毒素の切断箇所は標識されている。図1Bは、CFPおよびYFPが、CFPの励起によりYFPが蛍光発光するというFRETのためのドナー−受容体対として機能することを示している(上のパネル)。結合したCFPおよびYFP間のエネルギー転移は、ボツリヌス神経毒素によってシナプトブレビンあるいはSNAP-25フラグメントが切断された後に消失する(下のパネル)。CFPの最適励起波長は434nMであり、また、発光ピークについてはCFPで470nMであり、YFPは527nMである。
(発明の詳細な記述)
本発明は、特にリアルタイムでボツリヌス神経毒素を検出し、そしてそれら基質の切断活性をモニターするための、BoNTの基質であり毒素で切断されうるペプチドリンカーにより結合した蛍光プローブ間で起こる蛍光共鳴エネルギー転移(FRET)に基づいた、新しい複合体および方法を提供する。本発明の方法および複合体は、数時間内にピコモルレベルのBoNTの検出を可能とし、また、リアルタイムでの毒素の酵素動態を追跡することが可能である。該方法および複合体はさらに、培養細胞を用いた毒素阻害剤の大規模スクリーニングのための高処理能力の分析システムで用いることが可能であり、その毒素阻害剤には毒素の細胞への侵入および小胞膜を通じた移動に対する阻害剤を含む。本発明は生体細胞および神経細胞におけるボツリヌス神経毒素活性のモニターにも適している。
(例)
(材料および方法)
バイオセンサーDNA複合体の構築:pECFP-C1ベクター(Clontech社)をEcoRIおよびBamHI部位を用いてYFPcDNA(Clontech社)に挿入し、pECFP-YFPベクターを生成した。PCRを用いてラットsybIIのアミノ酸33-94をコードするcDNAを増幅し、XhoIおよびEcoRI部位(CFPとYFP遺伝子の間にある)でpECFP-YFPベクターに挿入して、形質転換細胞で用いることができるCFP-SybII-YFP(CFP-Syb(33-94)-YFPとも呼ばれる)複合体を生成した。CFP-SNAP-25-YFP(CFP-SNAP-25(141-206)-YFPとも呼ばれる)複合体も同様の方法で、SNAP-25の141-206残基を用いて作成した。また、リンカーとして完全長のラットSNAP-25Bを有する複合体(CFP-SNAP-25FL-YFP)も作成した。大腸菌を用いて組み換えキメラタンパク質を精製するために、NheIおよびBamHI部位を用いてpECFP-YFPベクターからCFP-SybII-YFP遺伝子およびCFP-SNAP-25-YFP遺伝子をpTrc-his(Invitrogen社)ベクターへ移動させた。
例1:CFP―YFP FRET対およびボツリヌス神経毒素プロテアーゼ活性に基づくバイオセンサー
ボツリヌス神経毒素プロテアーゼ活性を、FRET法を用いてモニターするために、sybIIまたはSNAP-25フラグメントを介してCFPとYFPタンパク質が結合され、そしてそれらは、それぞれCFP-SybII-YFPとCFP-SNAP-YFPとして示されている(図1A)。距離の増加とともに指数関数的に減少するCFP-YFPエネルギー転移効率を最適化するために、完全長のタンパク質のかわりに毒素基質の短いフラグメントが使われた。しかし、BoNTによる切断効率は標的タンパクが短くなりすぎると有意に減少する。したがって、シナプトブレビン配列のアミノ酸33-96残基を含む領域が使われた。なぜならその領域は、BoNT/B型、F型およびTeNTによる切断速度を完全長のシナプトブレビンと同様に保持することが報告されているからである。同様に、該複合体がBoNT/A型およびE型によって今までどおり認識され切断されうることを確実にするために、SNAP-25の141-206残基が選ばれた。
例2:バイオセンサータンパク質の切断の、ボツリヌス神経毒素によるインビトロでのモニタリング
300nMキメラタンパクCFP-SybII-YFPを、前還元された50nMのBoNT/B型ホロ毒素とキュベット中で混合した。BoNT/B型を添加後の様々な時点において(0、2、5、10、30、60分等)、発光スペクトルを得た。各スキャンの最後に、キュベットから少量の試料(30μl)を取り、SDSローディングバッファーと混合した。これらの試料を後にSDSページゲルにかけ、組換えキメラタンパク質でヒスチジン標識に対する抗体を用いて、キメラタンパク質の切断を可視化した。図3Aに示すように、バイオセンサータンパク質のBoNT/B型とのインキュベーションによって、YFP発光が減少しCFP発光が増加した。FRET比の減少は、BoNT/B型によるキメラタンパク質の切断の度合いと一致していた(図3A、下パネル)。この結果は、バイオセンサータンパク質の切断がそのFRET比の変化をリアルタイムで記録することによりモニターできるということを示している。
例3:マイクロプレート蛍光分光光度計を用いた、ボツリヌス神経毒素タンパク質活性のリアルタイムでのモニタリング
上記の実験により、ボツリヌス神経毒素の活性が、その標的バイオセンサータンパク質の発光スペクトルの変化をモニターすることにより、インビトロで検出できることが示された。次に我々は、バイオセンサータンパク質の切断をリアルタイムでマイクロプレート・リーダーを用いてモニターできるかを判断した。これは、この解析を将来のハイスループット・スクリーニングに適応できる可能性を示すことになる。図4Aに示すように、300nMのCFP-SNAP25-YFPキメラタンパク質を96ウェルプレート中でBoNT/A型と混合した。CFPは436nmで励起され、CFPチャンネル(470nM)とYFPチャンネル(527nM)の蛍光を30秒間隔で90分にわたり測定した。BoNT/A型の添加は、YFPチャンネル発光の減少とCFPチャンネル発光の増大を引き起こした。この結果により、複数の試料におけるボツリヌス神経毒素酵素活性のリアルタイムでの記録が可能となった。例えば、図4Bに示すように、様々な濃度のBoNT/A型またはE型が300nMのCFP-SNAP25-YFPに添加され、各試料のFRET比が図4Aに説明されるように同時にモニターされた。FRET比の変化は毒素の濃度に関連していた。つまり、毒素の濃度が高いとFRET比が速く減少した。このFRET比の変化は特異的である。なぜならCFP-SNAP-25-YFPのみ(図4Cの左パネル)または、CFPとYFPの混合物(図4Cの右パネル)のいずれにおいても有意な変化が検出されなかったからである。
例4:生細胞におけるボツリヌス毒素活性のモニタリング
CFP-YFPに基づくバイオセンサー解析は、インビトでのロボツリヌス神経毒素検出に使えるだけでなく、生細胞においても使うことができる。この応用を確立するために、PC12細胞にCFP-SNAP-25-YFPをトランスフェクトさせた。PC12細胞は、BoNT/A型およびE型を取り込むことができる神経内分泌細胞株である。トランスフェクトされた細胞はBoNT/A型(50nM)と72時間インキュベートされ、CFP-SNAP-25-YFPを発現した細胞のFRET比を、CFP-YFP FRETを検出するための特殊なフィルター・セットを装備したエピ蛍光顕微鏡で記録した。簡潔に説明すると、FRET比は二つのフィルター・セット、つまりCFP用(励起437nm/発光470nM)とFRET用(励起437nm/発光535nm)、を用いて取得した同じ細胞からの画像の蛍光強度の比として計算される。合計53個の細胞から集め、また同じバイオセンサータンパク質を発現しているが毒素にはさらされていない同数のコントロール細胞と比較した。図6Aに示されるように、BoNT/A型による72時間の処理により、実験した細胞群のFRET比が有意に減少した(p<1.47E-05)。野生型PC12細胞はBoNT/B型およびF型には影響されない。
例5 細胞に基づいたBoNTの検出
細胞に基づいた研究を実施するために、初めにCFP-SNAP-25(141-206)-YFPセンサーをPC12細胞にトランスフェクトした(図7a)。図2aに示すように、エピ蛍光顕微鏡とともに従来の3フィルター・セット方法を用いて、上の材料および方法の項目に記したように生細胞におけるFRETシグナルを得た(Gordonら、Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J. 74、2702-2713(1998);Sorkinら、Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Curr. Biol. 10、1395-1398(2000))。トランスフェクトされたPC12細胞を50nMのBoNT/A型で96時間処理した。それらの蛍光画像を分析し、標準化したFRET比(補正FRET/CFP)を図7bにプロットした。インビトロでSNAP-25(141-206)フラグメントが完全長のSNAP-25と同等の切断率を持つことが報告されているが(Washbourneら、Botulinum neurotoxin type A and E require the SNARE motif in SNAP-25 for proteolysis. FEBS Lett. 418、1-5(1997))、しかしながら、CFP-SNAP-25(141-206)-YFPは生細胞においては毒素基質として不十分であるようだった。BoNT/A型(50nM)との96時間のインキュベーションにより、細胞集団の中でFRET比の移動がわずかにしか(有意ではあるが)見られなかったため、切断が細胞中では非効率的であり、本センサーが細胞中での毒素検出のためには実用的でないことを示している。
Claims (10)
- 細胞原形質膜もしくは細胞小胞膜に結合することができる分子複合体をコードする単離ポリヌクレオチド分子であって、前記複合体が(1)リンカーペプチド、(2)蛍光ドナー部分、(3)蛍光受容部分、(4)細胞原形質膜もしくは細胞小胞膜に前記複合体を結合させるための膜結合ドメインを含むことと、前記リンカーペプチドがシナプトブレビン、シンタキシン及びSNAP−25からなるグループから選択されるボツリヌス神経毒素の切断認識部位を含むことと、前記膜結合ドメインが、配列7のパルミトイル化箇所(83−120残基)もしくは配列8の膜貫通ドメイン(95−116残基)を含むフラグメントを有することと、前記蛍光ドナー部分および前記蛍光受容部分が前記リンカーペプチドによってリンクされることとを特徴とする単離ポリヌクレオチド分子。
- 請求項1に記載のポリヌクレオチド分子を含み、プロモーターに動作可能にリンクされる発現ベクター。
- 前記プロモーターが誘導性プロモーターであることを特徴とする請求項2に記載の発現ベクター。
- 請求項1に記載の単離ポリヌクレオチド分子を含む細胞。
- 前記細胞が初代培養神経細胞、PC12細胞あるいはそれらの誘導体(derivative)、初代培養クロム親和細胞、神経芽腫細胞、アドレナリン作用性ヒトSK−N−SH細胞、およびNS−26細胞株からなるグループより選択されることを特徴とする請求項4に記載の細胞。
- 前記細胞が皮質神経細胞、海馬神経細胞、脊髄運動神経細胞、あるいはコリン作用性マウスNeuro 2a細胞であることを特徴とする請求項5に記載の細胞。
- 前記蛍光ドナー部分が緑色蛍光タンパク質(GFP)あるいはそれらの変異体であることと、前記蛍光受容部分が前記緑色蛍光タンパク質に対応する変異体であることとを特徴とする請求項1に記載の単離ポリヌクレオチド分子。
- 前記パルミトイル化箇所がCFP−SNAP−25(141−206)−YFPのN末端に融合されたことを特徴とする請求項1に記載の単離ポリヌクレオチド分子。
- 前記複合体がCFP−SNAP−25(FL)−YFP、YFP−Syb(FL)−CFPもしくはCFP−Syb(33−116)−YFPであり、前記SNAP−25(FL)が配列番号7で、Syb(FL)が配列番号8で、Syb(33−116)が配列番号10であることを特徴とする請求項1に記載の単離ポリヌクレオチド分子。
- 前記複合体が、そのN末端と融合した前記パルミトイル化箇所(83−120残基)を含むSNAP−25のフラグメントを有するCFP−SNAP−25(141−206)−YFPであって、前記SNAP−25(141−206)が配列番号9で、前記パルミトイル化箇所(83−120残基)が配列番号11であることを特徴する請求項1に記載の単離ポリヌクレオチド分子。
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US7208285B2 (en) * | 2001-08-28 | 2007-04-24 | Allergan, Inc. | Fret protease assays for botulinum serotype A/E toxins |
US6504006B1 (en) * | 2001-10-12 | 2003-01-07 | Nancy Rose Shine | Substrate peptides and assays for detecting and measuring proteolytic activity of serotype A neurotoxin from clostridium botulinum |
US7183066B2 (en) * | 2002-09-27 | 2007-02-27 | Allergan, Inc. | Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins |
JP5178009B2 (ja) | 2003-12-19 | 2013-04-10 | ウィスコンシン アルムニ リサーチ ファンデイション | ボツリヌス神経毒素検出のための方法および複合体 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2023239229A1 (ko) * | 2022-06-10 | 2023-12-14 | (주)메디톡스 | 보툴리눔 독소 안정화 조성물, 이를 포함하는 보툴리눔 독소 제제 및 이에 사용하기 위한 폴리펩티드 |
Also Published As
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EP2502930A3 (en) | 2012-10-10 |
US20120220490A1 (en) | 2012-08-30 |
US20160356776A1 (en) | 2016-12-08 |
CA2550401C (en) | 2015-12-08 |
EP2502930B1 (en) | 2016-06-29 |
CA2550401A1 (en) | 2005-08-25 |
US8137924B2 (en) | 2012-03-20 |
US8999649B2 (en) | 2015-04-07 |
WO2005076785A2 (en) | 2005-08-25 |
JP2007520211A (ja) | 2007-07-26 |
WO2005076785A3 (en) | 2009-03-19 |
EP2332959B1 (en) | 2014-11-26 |
EP1751284A2 (en) | 2007-02-14 |
IL176382A0 (en) | 2008-04-13 |
EP1751284A4 (en) | 2010-01-13 |
EP2332959A2 (en) | 2011-06-15 |
EP2332959A3 (en) | 2011-10-05 |
US20150253326A1 (en) | 2015-09-10 |
US20060134722A1 (en) | 2006-06-22 |
EP2502930A2 (en) | 2012-09-26 |
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