JP2004534534A - Genes encoding new molecular motor proteins and methods for diagnosing diseases related to the genes - Google Patents

Abstract

The present invention provides kIF1Bβ protein, a novel polypeptide having motor activity for transporting synaptic vesicle precursors. The invention also provides polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and uses thereof, for example, diagnostic methods for kiflBβ gene-related disorders. The polypeptide of the present invention, a gene encoding this polypeptide, and a compound that alleviates the symptoms of a Kif1Bβ gene-related disease develop a therapeutic or prophylactic agent for a kif1Bβ gene-related disease, for example, Charcot-Marie-Tooth disease type 2A. Useful to

Description

全てのデータは、平均±SDで示される。^＊p＜0.05、^＊＊p＜0.001、t検定
【００６８】
シナプスの構造を試験するために、本発明者らは、シナプス小胞密度を定量した。脊髄の後角をC4レベルで、従来の電子顕微鏡用に処理した。シナプスの60の領域を100nm厚の断片の電子顕微鏡写真から無作為に選択して、以前に記載されたとおり(Yonekawa, Y et al. (1998)J. Cell Biol. 141(2)、431-41)、4対の同腹仔について定量した。結果として、本発明者らは、神経末端のシナプス小胞の密度における有意な低下を見出した（図3Jまた図9Cも参照のこと）。
【００６９】
実施例３ KIF1Bβは、ニューロンにおいて自律的に細胞に作用する。
次いで、本発明者らは、kif1B^{＋ / ＋}、またはkif1B^-/-の海馬ニューロンを培養して、インビトロにおいてニューロン損失表現型を評価した。ニューロンをプレート後、5日連続して観察し、ニューロン分化について確立された基準(Goslin、K、およびBanker, G(1991)Rat hippocampal neurons in low-density culture. In Culturing Nerve Cells, G. Banker, and K. Goslin, ed. (Cambridge, MA；MIT Press), 251-82)を用いて、分化段階0〜4に分類して、定量した。以前に記載されたとおり(Goslin, K, and Banker, G(1991)Rat hippocampal neurons in low-density culture. In Culturing Nerve Cells, G. Banker, and K. Goslin ed. (Cambridge, MA；MIT Press), 251-82；Yonekawa、Y et al. (1998) J. Cell Biol. 141 (2), 431-41)、培養手順および定量的観察を実施した。図4A〜Bに示されるとおり、kif1B^-/-ニューロンの生存度は、2日後、有意に低下した。5日目まで、それぞれkif1B^-/-およびkif1B^{＋ / ＋}のニューロンの5％、10％、および90％が生存した（図4B）。ノックアウトニューロンを野生型グリアと同時培養した場合、本発明者らは、kif1Bが、細胞の生存度に対する寄与において、ニューロン中で自律的に細胞に作用すると結論した。
【００７０】
KIF1Bのどのアイソフォームがニューロン損失表現型についての原因である可能性が高いかを評価するため、本発明者らは、救済実験を企てた。EGFPタグ化アイソフォームをコードするアデノウイルスベクターを最初に用いて線維芽細胞を感染させて、イムノブロットによって発現を確認した（図4E）。アデノウイルス構築物の調製のために、KIF1BαおよびKIF1Bβの全長cDNAをEGFP（Clontech)を用いてC末端でタグ化し、そして以前に記載されたとおり（Nakata, T et al. (1998) J. Cell Biol. 140 (3), 659-74)、CAGプロモーターを有するpAdex-Wt1ベクター中に連結して、組み換えアデノウイルスを作製した。直径35mmの皿一枚あたり、1.0×10⁵個のニューロンをプレートし、プレートの日に、同じ力価のKIF1BαまたはKIF1Bβアデノウイルスを感染させ、5日後に生存率を観察した。感染効率は90％を超えており、そして感染したkif1B^{＋ / ＋}ニューロンの50〜80％が、5日目まで生存した。重要なことに、KIF1Bβを用いた感染（ただしKIF1Bα発現ウイルスではない）は、kif1B^-/-ニューロンの生存度を有意に救済した（図4C〜D）。KIF1Bαの欠失によって生じたミトコンドリア輸送における任意の潜在的な欠陥は、冗長なミトコンドリアモーター（例えば、従来のキネシンKIF5A、B、およびC：Tanaka, Y et al. (1998) Cell 93(7), 1147-58；Kanai, Y et al. (2000)J. Neurosci. 20(17)、6374-84)によって明らかに補償された。なぜなら、インビトロ（図11を参照）でも、インビボ（示さず）でも、ノックアウトマウスにおいて、ミトコンドリア分布に変化は観察されなかったからである。このように、KIF1Bβはおそらく、kif1B^-/-マウスにおけるニューロン欠失を担う主なアイソフォームであった。
【００７１】
実施例４ kif1B^{＋ /-}マウスにおける軸索輸送の特異的な障害
次いで、シナプス小胞前駆体の特定の輸送がモータータンパク質のハプロ不全によって障害されるか否かを試験するために、本発明者らはヘテロ接合体の末梢神経のタンパク質含量を評価した。研究のために、2ヶ月齢kif1B^{＋ /-}マウスの坐骨神経を選択した。なぜなら、それらは二次的な欠陥を発症し得る明白な表現型を示さなかったからである。本発明者らは、kif1B^{＋ / ＋}およびkif1B^{＋ /-}マウスの末梢神経におけるいくつかのタンパク質のレベルを、定常状態（図5A）および2時間の神経結紮による累積性の様式（図5B）において比較した。坐骨神経の結紮の2時間後、結紮した部位に対して近位または遠位で1cmの長さの部分を収集して、直ちに液体窒素中で凍結させ、プロテアーゼインヒビターを補充したRIPA緩衝液を含む乳鉢中で砕いて、標準的な方法(Hirokawa, N et al. (1990)J. Cell Biol. 111(3), 1027-37；Takeda, S et al. (2000)J. Cell Biol. 148(6)、1255-65)によってAP結合体化二次抗体を用いてイムノブロットのために処理した。kif1B^{＋ /-}マウスの坐骨神経におけるKIF1Bβタンパク質のレベルは、kif1B^{＋ / ＋}マウスにおけるレベルの半分であって、このことは、ハプロ不全の存在を支持した（図5A〜B）。シナプス小胞タンパク質に関して、シナプトタグミンおよびSV2のレベルは、野生型の約60％であったが、シナプス膜タンパク質SNAP-25およびシンタキシン、ミトコンドリアタンパク質COX I、またはコントロールのキネシンKIF5Aのレベルは、ヘテロ接合体において有意に変化しなかった（図5A）。神経結紮実験によってさらに、シナプス小胞タンパク質の輸送が特別に減少されたことが示された（図5B）。この実験において、ヘテロ接合型軸索において近位部位で蓄積されているシナプス小胞タンパク質の量は、野生型軸索における量に比べて低下していたが、近位部分に蓄積されているシナプス原形質膜タンパク質の量は、ヘテロ接合型と野生型軸索との間で変化はなかった。総タンパク質を染色しても、主な細胞骨格タンパク質のレベルにおいて明白な相違は示されなかった（図5D）。このように、シナプス小胞タンパク質輸送における低下は、軸索輸送の非特異的な全体的障害に起因する可能性は低かった。ニューロンの細胞体が位置する脊髄において、KIF1Bβのレベルのみが低下していたが、シナプトタグミンおよびSV2のレベルは低下しなかった（図5C）。これらのデータを考慮すると、シナプス小胞タンパク質のレベルは、神経軸索において低下したが、ヘテロ接合体の細胞体においては低下しなかったことが示唆され、このことはKIF1Bβモーターによって正常に作動された軸索輸送の特異的な障害を反映する。
【００７２】
実施例５ 慢性性末梢神経障害に罹患したkif1B^{＋ /-}マウス
kif1B^{＋ /-}マウスは、1年齢を超えると徐々に千鳥足になった。彼らは、最終的に自分の体重を支えることができなくなり、時折、跳ねることによってしか動けなくなった（図6A）。本発明者らは、慢性神経障害を疑い、以下の行動学的および生理学的試験によって2ヶ月齢、および12ヶ月齢のマウスを試験した。
【００７３】
kif1B^{＋ /-}マウスは、行動学的試験において、進行性の筋力低下、および運動調整不全(discoordination)を示した。本発明者らは、固定バー試験およびロータロッド試験を実施した。固定バー試験では、20mm幅の水平な木製のバーの上にマウスを置いた。3回訓練した後、マウスの保持時間は、以前に記載されたとおり、最大60秒と記録された(Kadotani, H et al. (1996)J. Neurosci. 16(24), 7859-67)。ロータロッド試験では、5分間に4rpmから40rpmに加速する回転ロッド上にマウスを置いた。そしてその保持時間を、本質的に以前に記載されたとおり測定した(Jones, B. J. 、およびRoberts, D. J. (1968)J. Pharm. Pharmacol. 20(4), 302-4)。5日間連続して1日あたり3回試行した。結果として、本発明者らは、12ヶ月齢のkif1B^{＋ /-}マウスについては、コントロールについてよりも有意に短い保持時間を検出した（表2）。固定バー試験において、12ヶ月齢のkif1B^{＋ /-}マウス（このマウスは、震えていた）は、コントロールよりも、落下する傾向が有意に高かった（表2）。ロータロッド試験において、ヘテロ接合体マウスはまた、訓練の連続する5日を通じて、有意な能力の障害を示した（図6B；表2）。12ヶ月齢のkif1B^{＋ /-}マウスの把握力は、kif1B^{＋ / ＋}マウスの把握力よりも20〜25％弱かった（表2）が、ホットプレート試験を用いて検査した感覚障害は有意ではなかった（データ示さず）。把握強度は、以前に記載されたように測定した(Meyer, O. A. et al. (1979)Neurobehav. Toxicol. 1(3)、233-6)。ホットプレート試験では、56℃のヒートブロックの上にマウスを置き、前肢および後肢をなめる(licking)までの潜時を、60秒のカットオフ潜時で記録した。
【００７４】
（表２） 動物の行動解析

データは、固定バー実験および把握力実験において平均±SEとして記し、電気生理学的実験では平均±SDとして記す。^＊p＜0.01（マン-ホイットニーのU検定）。^＊＊p＜0.0005（t検定）。^＊＊＊p＜0.05（マン-ホイットニーのU検定）。
【００７５】
筋力低下の性質を評価するため、本発明者らは、真ん中高めのレベルで、直接刺激後、足底筋で記録した12ヶ月齢の坐骨神経の誘発された複合活動電位を分析した（図6C）。末梢神経伝導速度を、以前に記載されたとおり(Robertson, A et al. (1993)Acta Neuropathol. (Berl)86(2)、163-71)（ただしわずかに改変して）、坐骨神経について決定した。遺伝子型を知らない状態で、全ての記録を実施した。エーテル麻酔下で、坐骨神経を見て、0.1ミリ秒の方形波の坐骨のノッチまたは真ん中高めのレベルで直接刺激し、後肢の足底筋において経皮的に刺入した電極を用いて、複合筋活動電位を記録した。伝導時間を刺激電極と記録電極との間の距離で割ることによって、運動神経伝導速度(MNCV)を算出した。結果として、振幅はkif1B^{＋ /-}マウスでは有意に低下したが、伝導速度は、なんら有意な相違を示さなかった（図6C〜D；表2）、このことは、神経の脱髄が要因ではないことを示唆した。さらに、遠位筋肉の組織学的分析は、有意な変化を示さなかった（データ示さず）。このように、神経軸索における欠陥が、kif1B^{＋ /-}マウスの末梢神経障害に最も有意に寄与する可能性が高い。
【００７６】
実施例６ ヒトKIF1B変異体は、CMT2A神経障害を起こす
最終的に、本発明者らは、CMT2A（II型シャルコー・マリー・ツース病の常染色体優性サブタイプ）が、ヒトにおけるKIF1Bに基づく神経障害であることを示した。本発明者らがこのことを疑った理由は、ヒト疾患に対するマウス表現型の類似性、およびKIF1Bが第一染色体上でマッピングしたCMT2Aの間隔に関連するからである。本発明者らは、CMT2Aの、以前に十分特徴付けられたヒトの系図（常染色体優性の遺伝的パターンを有する）を分析した（図7A；Saito, M et al. (1997)Neurology 49(6)、1630-5)。CMT2Aの家族＃694における12例の日本人個体由来の末梢血白血球から、高分子量DNAを調製した(Saito, M et al. (1997)Neurology 49(6)、1630-5)。ヒトKIF1B遺伝子の完全なエキソン-イントロン構造に基づいて(Yang, H. W. et al. (2001)Oncogene, 20(36)、5075-83)、各々の47個のエキソンを、記載されたとおりゲノムPCRによって増幅して(Yang, H. W. et al. (2001)Oncogene，20(36)、5075-83)、SSCPおよび／または直接シーケンス解析に供した。低pH緩衝系を用いてSSCP解析を実施して、より長いフラグメントを分離した(Kukita, Y et al. (1997)Hum. Mutat. 10(5), 400-7)。PCR産物を精製して、ABIシーケンサー(Perkin Elmer)を用いて直接配列決定した。多くの一塩基遺伝子多型(SNP)の中でも、本発明者らは、エキソン3中の異常なSSCP移動度シフトが、完全に臨床的兆候と関連していることを検出した。このことは、この系図の4例の罹患した人全部において検出されたが、8例の罹患していない兄弟姉妹でも、95例の関連のない日本人コントロールでも検出されなかった（図7B）。以下のプライマーセット、5'-TGGAAGCAATCAAGTAAGTATAGA-3'（配列番号6）、および5'-CCCCGCATAATGTTCAAGCC-3'（配列番号7）を用いて、95℃30秒、60℃30秒、および72℃30秒を35サイクルの条件下で、続いて72℃で10分の伸長によって、ヒトkif1Bβオーソログのエキソン3を増幅した。次いで、本発明者らは、PCR産物を直接配列決定して、ヘテロ接合性のA→Tの点突然変異（これが、罹患した個体においてQ⁹⁸をLに転換した）を明らかにした（図7C）。
【００７７】
このQ98L突然変異は、コンセンサスATP結合部位である、Pループの真ん中に存在しており、それによってこのメカノケミカル酵素の機能を破壊すると予測される。従って、本発明者らは、Q98L突然変異を用いて、組み換えマウスKIF1Bβのモーター活性を試験した。Q98L突然変異を、PCR増幅によって、全長マウスkif1Bβ cDNA中に導入して、配列決定によって確認した。最初に、以前に記載されたような標準的方法を用いて(Maeda, K et al. (1992)Mol. Biol. Cell 3(10), 1181-94)、本発明者らは、野生型およびQ98L組み換えKIF1Bβの全長タンパク質の微小管活性化ATP代謝回転を測定したが、これはそれぞれ3.6±0.5、および0.0±0.4nmol/mg/分であった。このように、突然変異されたモーターのATPase活性は有意に低下した。本発明者らは、さらに、Vero細胞において、Q98Lまたは野生型KIF1Bβを発現させて、インビボにおけるそれらの運動性を試験した。陽イオン性脂質を用いて、Vero細胞をトランスフェクトして、24時間培養し、そして抗KIF1Bβポリクローナル抗体および抗αチューブリンモノクローナル抗体DM1A(Sigma)を用いて免疫染色した。微小管の陽極末端に向かうその運動性に起因して、過剰発現した野生型KIF1Bβタンパク質は、細胞周辺に蓄積した（図7D、左側）。対照的に、Q98L変異体タンパク質は、核周囲の領域に残留して凝集した（図7D、右側）。これらのデータによって、Q98L点突然変異がモーター活性の機能的な損失を生じることが示唆された。
【００７８】
産業上の利用可能性
本発明は、シナプス小胞前駆体を輸送するモーター活性を有する新規なポリペプチドであるKIF1Bβタンパク質を提供する。本発明はまたこのポリペプチドをコードするポリヌクレオチド、このポリヌクレオチドを含むベクター、このベクターを保有する宿主細胞、このポリペプチドを調製するための方法、およびkif1Bβ遺伝子関連疾患を診断するための方法を提供する。さらに、本発明は、kif1Bβ遺伝子関連疾患の症状を緩和する化合物についてスクリーニングする方法を提供する。本発明のポリペプチド、このポリペプチドをコードする遺伝子、およびkif1Bβ遺伝子関連疾患の症状を緩和する化合物は、kif1Bβ遺伝子関連疾患、例えば、シャルコー・マリー・ツース病2A型のための治療剤または予防剤を開発するために有用である。
【図面の簡単な説明】
【００７９】
【図１】マウスKIF1Bβの分子クローニングおよび抗体特徴付けを示すダイアグラムおよび写真を示す。（A）kif1B cDNAの略図。DNAマトリックスプロット（ウインドウサイズ＝30、最小スコア％＝65）は、テールドメインにおけるスプライシングバリエーションを示す。（B）抗KIF1Bβ抗体でプローブした成体マウス組織のイムノブロット。（CおよびD）KIF1Bβに対する免疫蛍光顕微鏡検査法。（C）前角における運動ニューロン。（D）軸索路を示す、脊髄の断面。バーは、10μm。
【図２】マウスKIF1Bβとシナプス小胞前駆体の会合を示す写真である。（A）ナイコデンツ（Nycodenz）密度勾配フローテーションアッセイであって、これは、KIF1Bβがシナプトタグミンを含有する小胞画分に存在することを示す。矢印は、浮かんだ(floated)ピークを示す。（B）KIF1Bβ会合小胞のGSTプルダウンアッセイ。シナプス小胞タンパク質は、KIF1Bβテール領域(aa885-1700)に会合した小胞中で特異的に検出されたが、プレックストリン(pleckstrin)相同性(PH)ドメインを欠く短縮構築物(ΔPH；aa885-1528)に会合した小胞中では検出されなかった。（C）抗KIF1Bβ、およびいくつかの抗シナプス小胞タンパク質抗体を用いる、マウス脳の小胞画分の免疫沈降。コントロールには、KIF17を用いた。シナプス小胞タンパク質であるシナプトタグミン、シナプトフィジン、およびSV2を、KIF1Bβ、COXI、チトクロムオキシダーゼI（ミトコンドリアのマーカー）と同時免疫沈降した小胞中で特異的に検出した。（D）軸索小胞上のKIF1Bβ（10-nm金）、およびシナプス小胞タンパク質（5-nm金）の共存を示すイムノEM。バーは、100nm。無作為に選択した領域における、全ての小胞の数を上回る、両方のシグナルを担持する小胞の数の割合を示した。^＊＊、p＜0.001（独立性検定、カイ二乗）。
【図３】マウスkif1B遺伝子の標的化破壊を示すダイアグラムおよび写真を示す。（A）kif1Bノックアウトについてのターゲティングストラテジー。HincIIによるゲノムサザンブロットについてのストラテジーがまた示される。A＋T/pau、AT-リッチ休止(pausing)シグナル；pBS、pBluescriptII-SK(＋)；Hc、HincII。（B）ゲノムサザンブロットであって、ここでは、組み換え対立遺伝子は、2.1kbのさらなるバンドを与える。（C）それぞれ、抗KIF1Nβ抗体、および抗KIF1Bα抗体を用いる、18.5dpcのマウス脳からの粗抽出物のイムノブロット解析。両方の場合とも、kif1B^-/-サンプルからバンドは検出されなかった。（D）新生同腹仔の外観。kif1B^-/-仔で特異的に見出される、前肢の垂れ下がり（矢印）、および前弯性姿勢（矢尻型）に注目のこと。（E）HE染色した肺断面。肺胞（アスタリスク）は、コントロールマウスにおいては、空気で膨張させられただけであることに注意のこと。バーは、25μm。（F〜I）kif1B^-/-マウスにおける脳欠陥。（F〜G）18.5dpcでの、kif1B^{＋ / ＋}（F）、およびkif1B^-/-（G）マウスの延髄の断面における呼吸中枢の比較。MpD:背側延髄網状核、MdV:腹側延髄網状核、VLRt:腹外側網状核。バーは、500μm。（H〜I）18.5dpcでの、kif1B^{＋ / ＋}（H）、およびkif1B^-/-（I）の胚の大脳半球の前額断（交連線維の不完全形成を伴う）。矢尻型は、前交連；矢印は、内包。バーは、600μm。（J）18.5dpcでのkif1B^-/-マウスの脊髄の前角領域の神経末端におけるシナプス小胞の密度の低下。＃1〜＃4は、カウントした4つの異なる領域を示す。
【図４】kif1B^-/-海馬の初代培養における神経細胞死を示すダイアグラムおよび写真を示す。（A）各々の遺伝子型の初代培養の時間的経過。分化の有意な遅延を伴うkif1B^-/-ニューロンの重篤なニューロン死亡に注意のこと。矢印は、死亡した細胞。バーは、20μm。（B）各々の遺伝子型のプレートしたニューロンの生存曲線および分化曲線。1単位面積あたりの最初の細胞数を100％として、ニューロン細胞数の相対指数を算出した。（C〜E）救済実験である。（C）感染の5日目のkif1B^-/-海馬ニューロンの救済された培養物。緑色蛍光は、各々のアデノウイルスベクター（EGFPによってタグ化されたkif1Bアイソフォームを発現する）を発現するニューロンを示す。ニューロンは、kif1Bβ構築物のみを用いて高度に分化した段階に到達したことに注目のこと。矢印は、死亡した細胞。バーは、10μm。（D）kif1B^-/-ニューロンの救済効率の定量。^＊p＜0.001．βはkif1Bβ、αはkif1Bα、Nは、非感染コントロール。（E）抗GFP抗体を用いる、感染した線維芽細胞溶解物のイムノブロット。各々のEGFPタグ化タンパク質は、予期される分子量のバンドを生じた。
【図５】kif1B^{＋ /-}坐骨神経軸索におけるシナプス小胞タンパク質のレベルの低下を示す写真を示す。（A）坐骨神経抽出物のイムノブロット。各々のレーンに10μgのタンパク質をロードした。kif1B^{＋ /-}神経におけるタンパク質レベルは、kif1B^{＋ / ＋}神経におけるタンパク質レベルの割合として示された。（B）ライゲーションの2時間後の坐骨神経におけるプールされたオルガネラのイムノブロット。結紮された部位に対して1cmの領域近位(Prox）、および遠位(Dist）からのタンパク質レベルを、比較した。（C）（A）と同様に調製した脊髄抽出物のイムノブロット。（D）坐骨神経および脊髄由来の抽出物のクマーシーブリリアントブルーによるタンパク質染色。各々のレーンに10μgの総タンパク質をロードした。
【図６】kif1B^{＋ /-}マウスにおける慢性的末梢神経障害を示すダイアグラムおよび写真を示す。（A）1.5歳齢のマウスの代表的な姿勢。kif1B^{＋ /-}マウスは、自身の体重を支持できなかったことに注目のこと。（B）5つ連続するトライアルにおける2ヶ月齢および12ヶ月齢のマウスのロータ-ロッド試験における保持時間。5日間のトレーニング後の改善にかかわらず、12ヶ月齢のkif1B^{＋ /-}マウスの保持時間は、kif1B^{＋ / ＋}マウスの保持時間よりも依然として有意に短かった(n＝12、p＜0.005、ANOVA検定)。（C〜D）12ヶ月齢のマウスの坐骨神経の電気生理学的解析。（C）足底筋肉での誘発された活動電位の典型的トレース。（D）運動神経伝導速度(MNCV）、および誘発された筋電位の振幅の数値化。振幅は低下されたが、MNCVは、有意に変化しなかった。このことは、この疾患が軸索起源であることを示唆する。
【図７】CMT2A患者由来のヒトKIF1B遺伝子における、機能損失の変異を示すダイアグラムおよび写真を示す。（A）＃694ファミリーの系図。丸印は雌性、四角は雄性、斜線は死亡した個体、白抜きの図形は未影響、黒塗りの図形は影響されたもの。（B）P-ループエキソン3についてのPCR-SSCP解析における運動度シフト。矢印は、影響を受けた個体における、特別に観察されたシフトしたバンド。（C）P-ループ領域における配列のエレクトロフェログラム(electropherogram)。影響を受けた個体由来のサンプルにおいては、AおよびTは、変異したコドンで同時に読まれ、その結果、ヘテロ接合性のQ98L点突然変異が生じる。（D）野生型またはQ98L変異KIF1Bβ構築物のいずれかによってトランスフェクトされたVero細胞の免疫蛍光検査。赤および緑のシグナルは、それぞれKIF1Bβおよびチューブリンを示す。KIF1Bβタンパク質の蓄積された位置における相違（矢印）に注目のこと。バーは、15μm。
【図８】KIF1Bβの空間時間的な発現パターンについてのイムノブロットを示す写真を示す。（A）4週齢のマウス神経系におけるKIF1Bβの広範な分布。（B）脳におけるKIF1Bβの連続的な発生上の発現。20μgのタンパク質を各々のレーンにロードした。
【図９】kif1B^-/-マウスにおける新生児無呼吸表現型の中央起源（ただし末梢起源ではない）を示す写真を示す。（A）各々の遺伝子型の新生児の肺におけるII型肺胞上皮細胞の電子顕微鏡写真。分泌された層板体（矢印）が、両方のサンプル中で見出されることに注目のこと。このことは、肺上皮の機能が、kif1B^-/-マウスにおいて保存されることを示唆した。バーは2μmである。（B）各々の遺伝子型の新生児の肋間筋の電子顕微鏡写真。kif1B^-/-マウスにおける筋線維の正常な発達によって、この表現型が筋肉起源であることは除外される。バーは2μm。（C）脊髄の後角領域の電子顕微鏡写真であって、神経終末におけるシナプス小胞の密度がkif1B^-/-マウスにおいて有意に低下したことを示す。テキストを参照のこと。バーは、400nmである。
【図１０】kif1B^-/-マウスにおける顔面神経核の不完全な発達を示す写真を示す。（A）各々の遺伝子型の各々の段階での、延髄のHE染色した7μm厚のパラフィン包埋断面。顔面神経核（丸で囲んだ）のサイズは、kif1B^{＋ / ＋}胚において徐々に増大したが、kif1B^-/-胚においては有意に減少しており、このことは、インビボにおけるニューロンの発達および生存における、kif1B遺伝子の機能的な重要性を示す。バーは400μm。（B）各々の遺伝子型の18.5dpcの顔面神経核（丸囲み）のトルイジンブルー染色した7μm厚のパラフィン断面。細胞体の数の有意な減少に注目のこと。ニッスル小体の染色が弱いほど、kif1B^-/-ニューロンの生存度の低さを反映する。バーは80μm。
【図１１】Mitotracker(Molecular Probes)によってプローブされた各々の遺伝子型の培養された海馬ニューロンにおけるミトコンドリア染色を示す写真を示す。このミトコンドリアは、両方の表現型の神経軸索において同様に分布したことに注目されたい。バーは10μm。【Technical field】
[0001]
Technical field
The present invention relates to KIF1Bβ protein, a novel polypeptide having motor activity for transporting synaptic vesicle precursors. The present invention also relates to polynucleotides encoding the polypeptide, methods for preparing the polypeptide, and methods of using the peptide, such as methods for diagnosing a kiflBβ gene-related disease.
[Background Art]
[0002]
Background art
Axonal transport supplies the nerve endings with essential organelles and materials, primarily by using microtubules as pathways. The kinesin superfamily of molecular motor proteins (KIF) has over 40 members that potentially function in various aspects of axonal transport (Hirokawa, N (1998) Science 279, 519-26). All superfamily members have a similar motor domain, which may also be an evolution made by multiple gene duplications. The remainder of the molecule is largely branched, possibly associated with multiple classes of cargo molecules. Indeed, each motor functional specificity is supported by a series of knockout mice displaying a pronounced developmental and subcellular phenotype (Yonekawa, Y et al. (1998), J. Cell Biol. 141 (2), 431-41; Tanaka, Y et al. (1998) Cell 93 (7), 1147-58; Nonaka, S et al. (1998) Cell 95 (6), 829-37; Takeda, S et al. (1999) J. Cell Biol. 145 (4), 825-36), but this phenotype is only marginally compensated by other KIFs. Defects in an unknown isoform of KIF may be specifically associated with a neurological disorder. Thus, the identification and characterization of these isoforms results in the diagnosis, treatment, and prevention of such disorders.
[0003]
Charcot-Marie-Tooth disease (CMT), the most common normal hereditary peripheral neuropathy in humans, with a morbidity of 1/2500, weakens and atrophy peripheral muscles and suppresses deep tendon reflexes Or clinically characterized by absence or mild loss of sensation (Shy, ME et al. Ed. (1999) Charcot-Marie-Tooth Disorders. Ann. New York Acad. Sci. 883). By using motor nerve conduction velocity (MNCV) as a marker of myelin degeneration, the disease has been classified into type I (myelinating disorders) and type II (axonal disorders) (Harding, AE, and Thomas, PK (1980) J. Med. Genet. 17 (5), 329-36). CMT2A (autosomal dominant subtype of type II CMT) has been mapped to human chromosome 1p35-36 (Ben Othmane, K. B. et al. (1993) Genomics 17 (2), 370-5). Interestingly, the human Kif1B locus is closely related to the CMT2A interval (Gong, T. -WL et al. (1996) Mamm. Genome7 (10), 790-1; Nakagawa, T et al. (1997) Proc. Natl. Acad. Sci. USA 94 (18) 9654-9). However, the gene responsible for CMT2A has not yet been identified.
DISCLOSURE OF THE INVENTION
[0004]
Disclosure of the invention
An object of the present invention is to provide a novel polypeptide having a motor activity for transporting synaptic vesicle precursors. It is another object of the present invention to provide polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and uses thereof (eg, a method for diagnosing a kif1Bβ gene-related disease).
[0005]
The present inventors have identified a novel isoform of the conventional mitochondrial motor KIF1B, called KIF1Bβ (Nangaku, M et al. (1994) Cell 79 (7), 1209-20; hereinafter referred to as KIF1Bα. Identified and characterized. The two isoforms (vinegar-priced variants of the same gene located on mouse chromosome 4E) share an N-terminal motor domain but have a distinct tail domain. The tail domain of KIF1Bβ has much in common with the domain of KIF1A, a motor that transports synaptic vesicle precursors (Okada, Y et al. (1995) Cell 81 (5), 769-80). Since their tail domains do not share homology with each other, the cargo of KIF1Bα and KIF1Bβ is expected to be different (Nakagawa, T et al. (1997) Proc. Natl.Acad.Sci. USA 94 (18) 9654-9; Nakagawa, T et al. (1998) Mol. Biol. Cell 9, 3939). We show evidence that KIF1Bβ transports synaptic vesicle precursors and characterized its functional significance in vivo (indicating that knockout mice developed neurological disorders) .
[0006]
In addition, we have found that kif1B^{+ /-}We demonstrated that the mouse phenotype resembled the symptoms of Charcot-Marie-Tooth disease (CMT). Therefore, we analyzed the KIF1B locus in CMT2A patients. We have now discovered a Q98L missense mutation in the motor domain ATP binding consensus. We then showed that the Q98L mutant KIF1Bβ protein had reduced ATPase activity and motility. This suggests that haploinsufficiency of this motor protein is responsible for CMT2A neuropathy in both this mouse model and humans.
[0007]
The above results can be considered as follows.
[0008]
Here we have identified KIF1Bβ, a new motor protein that transports synaptic vesicle precursors. In addition, we have generated kiflB heterozygous mice that mimic human CMT2A neuropathy. Our evidence present here indicates that the autosomal dominant inheritance pattern of CMT2A can be explained by KIF1B haploinsufficiency. (1) kif1B^{+ /-}Mice reduced KIF1Bβ levels in half and developed tardive axonal degeneration similar to CMT2A. (2) Human KIF1Bβ is closely related to CMT2A. (3) The human Q98L mutant was completely associated with clinical signs. (4) The Q98L mutant disrupted the motor function of KIF1Bβ. (5) As a functional consequence, the level of cargo, a synaptic vesicle protein such as synaptotagmin and SV2, is increased by kif1B^{+ /-}It was specifically reduced in peripheral axons of mice.
[0009]
The functional significance of KIF1B is that kif1B died from apnea^-/-It was even more evident in newborn mice. Impaired development of the brain stem nucleus and commissural axons, and impaired synapse formation in the spinal cord,^{+ /-}It may be considered a more severe phenotype of the peripheral neuropathy seen in mice. kif1B^-/-Low neuronal survival (rescued by expressing KIF1Bβ cDNA but not by KIF1Bα) suggests that KIF1Bβ acts autonomously on cells in neurons, suggesting that axonal rather than myelin damage Consistent with the clinical definition of CMT2A as a disorder.
[0010]
Transport of synaptic vesicle precursors was also impaired in mice lacking the related motor, KIF1A (Yonekawa, Y et al. (1998) J. Cell Biol. 141 (2), 431-41). kif1A^-/-The mice also exhibited a lethal neurological phenotype during the neonatal period, suggesting a reduced density of synaptic vesicles at the nerve endings. But kif1B^-/-The mouse phenotype is kif1A^-/-It was more severe than the phenotype of mice (mice that could survive about 24 hours after birth). Neurological findings such as forepaw dropping, lordotic posture, and apnea are also important in kif1B^-/-Specific to newborn mice. Such a difference in severity appears in KIF1B at an early stage of development (FIG. 8), but KIF1A appears at a later stage of neurodevelopment (Yonekawa, Y et al. (1998) J. Cell Biol. 141 ( 2), 431-41). Although the patterns of expression of KIF1Bβ and KIF1A during development were different, they appeared to be functionally redundant in the same neurons due to the transport of synaptic vesicle precursors. Although KIF1A is neuron-specific, KIF1Bβ has a broad spectrum of tissue distribution, so that KIF1Bβ may also play a role in transporting other vesicles in non-neural tissues . Assessment of the genetic interaction between these two related genes is for further study.
[0011]
How could molecular motor deficiency produce the described neurological symptoms? Because axons lack a protein synthesis mechanism, proteins essential for neuronal survival must be transported to the axons by molecular motors. Thus, reduced levels of motor proteins fail to maintain axonal requirements. Defective transport of synaptic vesicle precursors or other unidentified cargo molecules (eg, neurotrophic factor receptors) due to inactivation of KIF1B is likely due to impaired nerve endings (electron microscopy and electrophysiological analysis). As well as by reduced neuronal survival in knockout mice). The traditional distal-first theory of axonal neuropathy, which states that distal muscles are affected earlier and more severely than proximal muscles, This can be explained in part by the notion that it requires higher levels of motor molecules. Thus, the present inventors propose here a new disease entity of "motor proteinopathy" which represents a paradigm for motor protein-derived pathogenesis of axonal neuropathy.
[0012]
The present invention relates to kif1Bβ protein, a novel polypeptide having motor activity for transporting synaptic vesicle precursors. The present invention also relates to polynucleotides encoding the polypeptide, methods of preparing the polypeptide, and uses thereof, for example, methods of diagnosing a kif1Bβ gene-related disease. More specifically, the present invention relates to the following.
[1] A substantially pure polypeptide selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;
(B) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein one or more amino acids have been substituted, deleted, inserted, and / or added, and the amino acid sequence of SEQ ID NO: 2 A polypeptide that is functionally equivalent to the polypeptide comprising;
(C) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 A polypeptide that is functionally equivalent to a polypeptide.
[2] An isolated polynucleotide encoding the polypeptide according to [1].
[3] A vector comprising the polynucleotide according to [2].
[4] A host cell having the polynucleotide according to [2] or the vector according to [3].
[5] A polynucleotide that is complementary to the polynucleotide of [2] or a complementary strand thereof and contains at least 15 nucleotides.
[6] A method for producing the polypeptide according to [1], comprising the following steps:
(A) culturing the host cell according to [4];
(B) allowing the host cell to express the polypeptide; and
(C) collecting the expressed polypeptide;
[7] An antibody that binds to the polypeptide of [1].
[8] An antisense polynucleotide to the polynucleotide according to [2].
[9] A transgenic non-human vertebrate, which has an exogenous polynucleotide encoding the polypeptide according to [1] in an expressible manner.
[10] A transgenic non-human vertebrate, wherein the expression of an endogenous polynucleotide encoding the polypeptide according to [1] is suppressed.
[11] The transgenic non-human vertebrate of [9] or [10], wherein the transgenic non-human vertebrate is a mouse.
[12] A method for diagnosing a kif1Bβ gene-related disease, the method comprising:
(A) detecting whether the subject has a mutation in a gene located in the same allele as the allele in which the gene encoding the nucleotide sequence of SEQ ID NO: 1 is located;
And wherein the subject is suspected of having a kif1Bβ gene-related disease if the subject has the mutation.
[13] A method for diagnosing a kif1Bβ gene-related disease, the method comprising:
(A) detecting the expression level of the subject's gene, wherein the gene is located at the same as the allele at which the gene encoding the nucleotide sequence of SEQ ID NO: 1 is located;
And a method in which the subject is presumed to have a kif1Bβ gene-related disease when abnormal expression is detected.
[14] The method according to [12] or [13], wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A.
[15] A method for screening for a compound that binds to the polypeptide according to [1], wherein the method comprises:
(A) contacting the test compound with the polypeptide according to [1];
(B) detecting the binding activity between the polypeptide and the test compound; and
(C) selecting a compound that binds to the polypeptide
A method comprising:
[16] A method of screening for a candidate compound for treating, alleviating, or preventing a kif1Bβ gene-related disease, the method comprising:
(A) administering a candidate compound to the transgenic non-human vertebrate according to any one of [9] to [11];
(B) observing changes in the symptoms of the kif1Bβ gene-related disease in the transgenic non-human vertebrate; and
(C) selecting a candidate compound that alleviates the symptoms of the kif1Bβ gene-related disease,
A method comprising:
[17] The method according to [16], wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A.
[18] A composition for treating, alleviating or preventing a kif1Bβ gene-related disease, wherein the composition is selected as the active ingredient by the method according to any one of [15] to [17]. A composition comprising a pharmaceutically effective amount of a compound and a pharmaceutically acceptable carrier.
[19] The composition according to [18], wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A.
[20] A method for treating, alleviating, or preventing a kif1Bβ gene-related disease, the method comprising pharmaceutically administering a compound selected by the method according to any one of [15] to [17]. A method comprising administering an effective amount.
[21] The method according to [20], wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A.
[0013]
The present invention relates to a novel protein named KIF1Bβ, a new motor protein that transports synaptic vesicle precursors. The nucleotide sequence of the isolated KIF1Bβ cDNA encompassed by the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the KIF1Bβ protein encoded by this cDNA is shown in SEQ ID NO: 2.
[0014]
For a given polypeptide as used herein, the term "substantially pure" means that the polypeptide is substantially free of other biological macromolecules. A substantially pure polypeptide is at least 75% (eg, at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method (eg, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis).
[0015]
Polypeptides that are structurally similar to the mouse KIF1Bβ protein are also included in the invention, as long as they have motor activity to transport synaptic vesicle precursors. Such structurally similar polypeptides also include variants of the KIF1Bβ protein from other organisms.
[0016]
Such polypeptides can be prepared by those skilled in the art using, for example, standard mutagenesis methods. Methods for modifying amino acids in proteins known to those skilled in the art include, for example, a deletion-mutant preparation method (Kowalski.D. Et al., 1976, J. Biochem., 15, 4457; McCutchan, TF et al., 1984, Science, 225, 626-628), Kunkel method (kunkel, TA, 1985, Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel, TA et al., 1987, Methods Enzymol. 154: 367 -382), Gapped-duplex method (Kramer, W. and Fritz, H.-J., 1987, Methods Enzymol. 154: 350-367; Zoller, MJ and Smith, M., 1983, Methods Enzymol. 100: 468). -500; Susumu Hirose, 1991, Masami Muramatsu and Hiroto Okayama, ed., Experimental Medicine Separate Volume, Genetic Engineering Handbook, Yodosha, pp246-252), PCR (Masami Muramatsu, lab manual genetic engineering, 3rd edition, Maruzen Co., Ltd.) , 1996, pp.227-230), site-directed mutagenesis methods such as the cassette mutation method (Toshimitsu Kishimoto, 1991, edited by Masami Muramatsu and Hiroto Okayama, Handbook of Experimental Medicine, Genetic Engineering Handbook, Yodosha, pp253-260), etc. Is mentioned. For example, Transformer (trademark) Site-Directed Mutagenesis Kit (CLONTECH # K1600-1) can be used.
[0017]
The artificial modification of the amino acid in the polypeptide is usually within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids. Amino acid mutations in polypeptides can also occur naturally. As long as it has a motor activity for transporting synaptic vesicle precursors, a naturally occurring mouse "KIF1Bβ" protein (SEQ ID NO: 2) by artificial or natural amino acid substitution, deletion, addition, and / or insertion The present invention also includes polypeptides having different amino acid sequences.
[0018]
It is considered that the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as non-polar amino acids, and thus are considered to have properties similar to each other. In addition, examples of the non-charger include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. In addition, examples of acidic amino acids include Asp and Glu. Examples of the basic amino acid include Lys, Arg, and His. The β-branched chain amino acids include Thr, Val, and Ile, and the aromatic amino acids include Tyr, Phe, Trp, and His.
[0019]
The polypeptide formed by adding amino acid residues to mouse KIF1Bβ protein is composed of mouse KIF1Bβ protein and other peptides.
[0020]
In addition, the preparation of a protein structurally similar to the mouse “KIF1Bβ” protein, which has a motor activity for transferring synaptic vesicle precursors, can be performed by a known hybridization technique (Cell Engineering Annex 8, New Cell Engineering Experimental Protocol, 1991, Shujunsha, pp. 188-193 and 79-87; Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), 8.46-8.52) and polymerase chain reaction technology (Cell Engineering Supplement 8, New Cell Engineering Experiment Protocol, 1991, Shujunsha, pp. 171-186; Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), 14.1-14.35). That is, for those skilled in the art, a mouse “KIF1Bβ” cDNA sequence (SEQ ID NO: 1) or a part thereof is used as a probe, and an oligonucleotide that specifically hybridizes to mouse “KIF1Bβ” cDNA (SEQ ID NO: 1) It has become common practice to isolate DNA highly homologous to the mouse `` KIF1Bβ '' cDNA from various other organisms, and to obtain a protein structurally similar to the mouse `` KIF1Bβ '' protein from the isolated DNA. I have.
[0021]
The present invention includes a polypeptide encoded by a polynucleotide that hybridizes with a mouse “KIF1Bβ” cDNA as long as it has a motor activity of transferring a synaptic vesicle precursor. Other organisms from which such polypeptides are isolated include, but are not limited to, humans, monkeys, rats, rabbits, goats, cows, pigs, and the like. Polynucleotides encoding such polypeptides can be isolated, for example, from neurons, brain, and cultured neurons.
[0022]
A polynucleotide encoding a “KIF1Bβ” polypeptide derived from an organism other than a mouse usually has high homology to the nucleotide sequence of the mouse “KIF1Bβ” cDNA (SEQ ID NO: 1). "Highly identical" refers to sequence identity of at least 60% or more, preferably 80% or more, more preferably 90% or more at the amino acid level. As used herein, the "percent identity" of two amino acid sequences, or two nucleic acids, refers to Karlin, and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990), modified using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). BLAST nucleotide studies are performed using the NBLAST program (score = 100, wordlength = 12). By using the FASTA program, the BLAST program, and the like, protein homology search can be easily performed, for example, in the DNA Databank of Japan (DDBJ; National Institute of Genetics: Yata 1111, Mishima, Shizuoka 411, Japan). . BLAST protein searches are performed using the XBLAST program (score = 50, wordlength = 3). If there is a gap between the two sequences, gapped BLAST, as described in Altsuchl et al., Is used (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) are used.
[0023]
Hybridization conditions for isolating a polynucleotide encoding a polypeptide functionally equivalent to the mouse “KIF1Bβ” protein can be appropriately selected by those skilled in the art. As an example, in a hybridization solution containing 25% formamide (50% formamide under stringent conditions), 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA, After prehybridization at 42 ° C overnight, hybridization is performed by adding a labeled probe and keeping the temperature at 42 ° C overnight. Subsequent washing is performed at room temperature, 2 × SSC, 0.1% SDS (under stringent conditions, 50 ° C., 0.5 × SSC, 0.1% SDS). However, a plurality of factors such as temperature, formamide concentration, and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize similar stringency by appropriately selecting these factors. (Cell Engineering Supplement 8, New Cell Engineering Experiment Protocol).
[0024]
The polypeptide of the present invention can be prepared as a recombinant polypeptide using a genetic recombination technique in addition to a natural polypeptide. The natural polypeptide is obtained, for example, by subjecting an extract of a tissue (eg, a neuron and a brain) in which the “KIF1Bβ” protein is expressed to affinity chromatography using an antibody against the “KIF1Bβ” protein described below. Can be prepared. On the other hand, a recombinant polypeptide can be prepared by culturing cells transformed with a polynucleotide encoding the “KIF1Bβ” protein and recovering the polypeptide as described below.
[0025]
Examples of the partial peptide of the present invention include a partial peptide in the N-terminal region and a partial peptide in the C-terminal region of a protein from which the signal sequence has been removed, and the peptide can be used for preparing an antibody. . The partial polypeptide having an amino acid sequence specific to the protein of the present invention has a chain length of at least 7 amino acids, preferably at least 8 amino acids, more preferably at least 9 amino acids. The partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.
[0026]
The present invention also relates to a polynucleotide encoding the polypeptide of the present invention. The polynucleotide encoding the polypeptide of the present invention is not particularly limited as long as it can encode these polypeptides, and includes cDNA, genomic DNA, and synthetic DNA. In addition, all DNAs having a desired base sequence based on the degeneracy of the genetic code are included as long as they can encode the protein of the present invention.
[0027]
As used herein, an `` isolated polynucleotide '' is a polynucleotide, the structure of which is relative to the structure of any naturally occurring polynucleotide or greater than three. A polynucleotide that is not identical to the structure of any fragment of a naturally occurring genomic polynucleotide that spans separate genes. Thus, the term includes, for example: (A) DNA, which has part of the sequence of a naturally occurring genomic DNA molecule in the genome of the naturally occurring organism; (b) the resulting molecule is any naturally occurring A polynucleotide incorporated into a vector or into a prokaryotic or eukaryotic genomic DNA in a manner that is not identical to the vector or genomic DNA present; (c) cDNA, genomic fragments, polymerase chain reaction ( Fragments produced by PCR), or separate molecules, such as restriction fragments; and (d) a recombinant nucleotide sequence that is part of a hybrid gene (ie, a gene encoding a fusion polypeptide). Specifically excluded from this definition are polynucleotides of DNA molecules that are present in different mixtures of the following: (I) DNA molecules, (ii) transfected cells, or (iii) cell clones (eg, those present in a DNA library such as a cDNA or genomic DNA library).
[0028]
Accordingly, in one aspect, the invention provides an isolated polynucleotide encoding a polypeptide described herein, or a fragment thereof. Preferably, the isolated polynucleotide comprises a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 1. More preferably, the isolated nucleic acid molecule has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, relative to the nucleotide sequence set forth in SEQ ID NO: 1. 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In the case of an isolated polynucleotide that is longer or equivalent in length to a reference sequence, eg, SEQ ID NO: 1, a comparison is made using the entire length of the reference sequence. If the isolated polynucleotide is shorter than the reference sequence, for example, shorter than SEQ ID NO: 1, a segment of the reference sequence of the same length (exclude any necessary loops by homology calculation) A comparison is made.
[0029]
The cDNA encoding the polypeptide of the present invention includes, for example, the cDNA of SEQ ID NO: 1 or a fragment thereof, RNA complementary thereto, or a synthetic oligonucleotide containing a part of the sequence of the cDNA.<sup>32</sup>Screening can be performed by labeling with P or the like and hybridizing to a cDNA library derived from a tissue (eg, neuron or brain) expressing the polypeptide of the present invention. Alternatively, oligonucleotides corresponding to the nucleotide sequences of these cDNAs can be synthesized, amplified by polymerase chain reaction using cDNA derived from an appropriate tissue (for example, neuron or brain) as a template, and cloned. Genomic DNA includes, for example, cDNA described in SEQ ID NO: 1 or a fragment thereof, RNA complementary thereto, or a synthetic oligonucleotide containing a part of the sequence of the cDNA.<sup>32</sup>Screening can be performed by labeling with P or the like and hybridizing to a genomic DNA library. Alternatively, oligonucleotides corresponding to the nucleotide sequences of these cDNAs can be synthesized, amplified by the polymerase chain reaction using genomic DNA as a template, and the cDNA encoding the polypeptide of the present invention can be cloned. On the other hand, a synthetic DNA can be prepared, for example, by chemically synthesizing an oligonucleotide having a partial sequence of the cDNA shown in SEQ ID NO: 1, annealing it to make it double-stranded, and binding it with DNA ligase.
[0030]
These DNAs are useful for producing a recombinant polypeptide. That is, a transformant obtained by inserting a polynucleotide encoding the polypeptide of the present invention (for example, the polynucleotide set forth in SEQ ID NO: 1) into an appropriate expression vector and introducing the vector into appropriate cells is used. The protein of the present invention can be prepared as a recombinant protein by culturing and purifying the expressed protein. Since the polypeptide of the present invention is a secretory protein, it can be prepared, for example, by expressing it in mammalian cells and secreting it extracellularly.
[0031]
Specifically, for example, vectors for expression in Escherichia coli include pKK223-3, pKK233-2, pJLA502, and the like. Also, for example, it can be expressed as a fusion protein with another protein. Vectors for that purpose include, for example, pRIT2T, pGEX-2T, pGEX-3X and the like. These fusion proteins can be easily recovered using an affinity column. If a vector having a thrombin cleavage site or a factor Xa cleavage site between the desired polypeptide of the fusion protein and the partner polypeptide is used, only the target protein can be cut out. Examples of vectors for secreting proteins extracellularly or into the periplasm include pKT280 and pRIT5 (edited by Masato Okada and Kaoru Miyazaki, invincible Biotechnical Series Protein Experiment Notes on Extraction, Separation and Purification, Yodoseki Publishing, 1996, pp.139-149).
[0032]
In addition, the polypeptide of the present invention can be expressed in insect cells and mammalian cells using baculovirus. A baculovirus vector for mammalian cells includes, for example, pAcCAGMCS1 (edited by Masami Muramatsu, Lab Manual Genetic Engineering 3rd Edition, Maruzen Co., 1996, pp. 242-246).
[0033]
The recombinant polypeptide expressed in the host cell can be purified by a known method. When the polypeptide of the present invention is expressed in the form of a fusion protein in which a tag of a histidine residue, glutathione S-transferase (GST) or the like is bound to the N-terminus, a nickel column or a glutathione sepharose column may be used. It can be purified.
[0034]
The polynucleotide encoding the polypeptide of the present invention can also be applied to gene therapy for a disease caused by the mutation. Examples of a vector for use in gene therapy include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a lentivirus vector, a herpes virus vector, an alphavirus vector, an EB virus vector, a papilloma virus vector, and a foamy virus. And non-viral vectors such as cationic liposomes, ligand DNA complexes, and gene guns (Y. Niitsu, M. Takahashi, Molecular Medicine, Vol 35, No. 11, 1385-1395, 1998). . Gene transfer can be in vivo or ex vivo. It is also conceivable to co-introduce with another cytokine gene.
[0035]
The present invention also relates to a DNA that specifically hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and has a chain length of at least 15 bases. "Specifically hybridize" means that cross-hybridization with a polynucleotide encoding another protein does not significantly occur under the normal hybridization conditions described above, preferably under stringent hybridization conditions. Means Such DNA includes a probe or primer capable of specifically hybridizing with a polynucleotide encoding the polypeptide of the present invention or a polynucleotide complementary to the polynucleotide, a nucleotide or a nucleotide derivative (for example, antisense oligonucleotide and Ribozymes).
[0036]
The cDNA encoding the protein of the present invention or an oligonucleotide containing a part of its sequence can be used for cloning of the gene or cDNA encoding the protein of the present invention, or for amplification by PCR. Furthermore, it can be used for detection of gene or cDNA polymorphism or abnormality (gene diagnosis, etc.) by methods such as restriction fragment length polymorphism (RFLP) and single-stranded DNA higher-order structural polymorphism (SSCP).
[0037]
The present invention also relates to an antibody that binds to the protein of the present invention. The antibodies of the present invention include both polyclonal and monoclonal antibodies. Polyclonal antibodies can be obtained, for example, according to the method described in the document `` The University of Tokyo Institute of Medical Science, Cancer Research Division, Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol, Published in 1993, Shujunsha, pp. 202-217 '' Can be made. For example, the protein of the present invention purified from immunized animals such as rabbits, guinea pigs, mice and chickens, a partial peptide thereof, or a peptide synthesized based on the amino acid sequence of the protein of the present invention, is injected into the vein of the ear, in the groin, Inject into the back of the nail of the limb. Administration of the antigenic polypeptide may be performed with Freund's complete adjuvant or Freund's incomplete adjuvant. The antigen is usually administered every few weeks, and the booster can raise the antibody titer. Blood is collected periodically, an increase in antibody titer is confirmed by ELISA or the like, and after the final immunization, blood is collected from the immunized animal to obtain an antiserum. The IgG fraction can be purified from the antiserum by salting out, ion exchange chromatography, HPLC, or the like. Further, the antibody can be further purified by affinity chromatography using the immobilized antigen.
[0038]
On the other hand, monoclonal antibodies can be prepared, for example, according to the method described in the document `` Takao Koike and Katsushi Taniguchi, 1991, Masami Muramatsu and Hiroto Okayama, Ed. it can. Specifically, the immunized animal is immunized with the protein of the present invention or its partial peptide in the same manner as described above, and after the final immunization, the spleen or lymph node is collected from the immunized animal. The antibody-producing cells and myeloma cells contained in the spleen or lymph node are fused using polyethylene glycol or the like to prepare a hybridoma. The target hybridoma is screened, cultured, and a monoclonal antibody can be prepared from the culture supernatant. For purification of the monoclonal antibody, for example, an IgG fraction can be purified by salting out, ion exchange chromatography, HPLC, or the like. Further, the antibody can be further purified by affinity chromatography using the immobilized antigen.
[0039]
The antibody thus prepared is used for affinity purification of the polypeptide of the present invention. Further, it can be used for examination and diagnosis of diseases caused by abnormal expression or structural abnormality of the polypeptide of the present invention, detection of the expression level of the protein of the present invention, and the like. For example, a polypeptide can be extracted from tissue, blood, cells, or the like, and examined or diagnosed for abnormal expression or structure of the polypeptide of the present invention by Western blotting, immunoprecipitation, ELISA, or the like. It is also conceivable to use the antibody of the present invention for antibody therapy. When used for antibody therapy, human antibodies or human antibodies are preferred.
[0040]
The humanized monoclonal antibody can be used, for example, according to the literature “Hidekazu Isogai, 1988, Experimental Medicine Vol. 6, No. 10, 55-60”. Further, as described in the document “T. Tsunenari et al., 1996, Anticancer Res., 16, 2537-2544”, it can also be prepared using a molecular biological technique. In addition, human antibodies can be prepared by immunizing a mouse whose immune system has been replaced with a human with the protein of the present invention.
[0041]
The present invention also relates to a transgenic non-human vertebrate which carries the DNA of the present invention exogenously. Such animals include animals expressing a foreign polynucleotide and animals capable of inducing the expression of the foreign polynucleotide. Expression may be systemic or cell, tissue, or organ specific. Further, it may be time-specific. Further, for example, the expression may be induced by an external stimulus, or the expression may be modified (induced or suppressed) in the progeny by crossing.
[0042]
The transgenic non-human vertebrate of the present invention can be produced, for example, by introducing a vector expressing the polynucleotide of the present invention into a fertilized egg. The introduction of the polynucleotide can be carried out by mixing the vector and the egg and treating with calcium phosphate, or by a microinjection method under an inverted microscope or the like. Alternatively, the vector of the present invention may be introduced into embryonic stem cells (ES cells), and the selected ES cells may be introduced into fertilized eggs (pulmonary blastocysts) by microinjection (Gordon, JW et al, Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384, 1980). The obtained fertilized egg can be transplanted into the oviduct of a pseudopregnant recipient by mating with a vasectomy-ligated male mouse to obtain a litter. Prepare DNA from tail of offspring and confirm the retention of introduced DNA by PCR (Matsuya Katsuki, Ed., Manual of Developmental Engineering, Kodansha (1989); Japanese Society of Biochemistry, Ed. Chemistry Doujin (1991); Hogan, B. et al., 1986, "Manipulating the mouse embryo", in "A laboratory manual", Cold Spring Harbor laboratory press). From a chimeric animal in which a gene has been introduced into the germ line, a heterozygote can be obtained by crossing with a normal animal. A homozygote can be obtained by crossing heterozygotes.
[0043]
As a promoter used for expressing the polypeptide of the present invention in vivo, for example, a systemically expressed promoter, and other tissue-specific, time-specific promoters can be used (Sauer, B., 1998, Methods 14: 381-92).
[0044]
When producing a transgenic animal that expresses the polynucleotide of the present invention in a site-specific or time-specific manner, a Cre-loxP system or a yeast FLP system can be used. For example, a transgenic animal having a Cre recombinase gene downstream of a site-specific or time-specific promoter is prepared, and a transgene holding a vector separately linked to a DNA encoding the polypeptide of the present invention downstream of a general-purpose promoter is prepared. Create a transgenic animal. At this time, a stop codon or a transcription termination signal sandwiched by loxP is inserted between the promoter and the DNA encoding the polypeptide of the present invention. By multiplying two individuals, the polypeptide of the present invention can be expressed along with the expression of Cre (Sauer, B., 1998, Methods 14: 381-92).
[0045]
In addition, the transgenic non-human vertebrate of the present invention has at least a part of a polynucleotide encoding the endogenous protein of the present invention substituted, deleted, added and / or inserted, so that A transgenic non-human vertebrate whose expression is suppressed is included. Such a transgenic animal can be produced, for example, by gene targeting. Such transgenic non-human vertebrates include, for example,
(A) preparing a vector in which at least a part of a genomic DNA encoding the protein of the present invention is mutated by substitution, deletion, addition and / or insertion,
(B) introducing the vector into mammalian embryonic stem cells,
(C) selecting, from the cells obtained in step (b), cells in which the vector has been introduced into the genome by homologous recombination,
(D) preparing an embryo of the mammal;
(E) injecting the cells obtained in step (c) into the embryo obtained in step (d),
(F) developing the embryo obtained in step (e),
(G) selecting, from the individuals in which the vector has occurred, selecting an individual that retains cells in which the vector has been introduced into the genome by homologous recombination.
[0046]
A specific gene targeting method can be performed, for example, by the method described in the literature (Capecchi, M.R. (1989) Science 244, 1288-1292).
[0047]
In addition, a transgenic non-human vertebrate in which the expression of a DNA encoding the endogenous protein of the present invention is suppressed can also be prepared by using an antisense method. In the antisense method, (1) a vector containing a DNA encoding an RNA complementary to a transcription product of a polynucleotide encoding the polypeptide of the present invention, introduced into mammalian embryonic stem cells as described above, (2) This can be injected into a mammalian embryo, and (3) an individual can be obtained from the embryo. The administration of the vector was performed by intraventricular administration (Kagiyama, S. et al. (1999) Brain Res. 829, 120-124) and local administration to tumors (Rubenstein, M. et al. (1996) J. Surg. Oncol. 62, 194-200) and intravenous administration (Zhao, Q. et al. (1998) Antisense Nucleic Acid Drug Dev. 8, 451-458).
[0048]
The transgenic non-human vertebrate of the present invention is particularly useful as a model for a kif1Bβ gene-related disease (eg, Charcot-Marie-Tooth disease type 2A). These transgenic non-human vertebrates can be used for molecular analysis of kif1Bβ gene-related diseases and for development of therapeutic or prophylactic drugs for kif1Bβ gene-related diseases. For this transgenic non-human vertebrate of the present invention, mammals, particularly rodents (eg, mice and rats) are preferably used.
[0049]
The tests and diagnoses of the present invention can be performed by detecting structural variations or abnormal expression of the kif1Bβ gene. For example, the structural variation of the kif1Bβ gene can be detected by amplifying the kif1Bβ gene from genomic DNA or mRNA from a subject by PCR or the like, and determining the nucleotide sequence thereof. By comparing to the nucleotide sequence of a normal gene, the presence or absence of the mutation and the type of the mutation are determined. Here, the mutation includes a polymorphism. That is, detection of SNP (single nucleotide gene polymorphism) in the kif1Bβ gene may enable testing and diagnosis of the risk of developing a kif1Bβ gene-related disease (eg, Charcot-Marie-Tooth disease type 2A) and prognosis. Mutations in the kif1Bβ gene that attenuate motor activity (eg, ATPase activity of the KIF1Bβ protein, and cell motility in vivo) are determined to be risk factors for the development and progression of a kif1Bβ gene-related disease. Also, to detect abnormal expression of the kif1Bβ gene, for example, expression of the kif1Bβ gene in neurons from patients can be determined by microarray analysis, quantitative RT-PCR or semi-quantitative RT-PCR, immunohistochemical staining, Western blotting. It is analyzed by blotting or the like. Abnormal expression is determined by comparison to expression in normal tissue. Abnormal expression includes increased, decreased, and eliminated expression. A decrease in kif1Bβ gene expression is determined to be a risk factor for the development and progression of a kif1Bβ gene-related disease. The tests and diagnoses of the present invention include risk tests performed before the onset of the kif1Bβ gene-related disease, and classifications performed after the onset of the kif1Bβ gene-related disease, tests for creating a treatment plan, and the like. . For example, after the onset of a kif1Bβ gene-related disease, structural variations or abnormal expression of the kif1Bβ gene are detected by examining neural tissue, and the presence and type of the variations and abnormalities are determined. In addition, if pre-onset testing determines a high risk for a kif1Bβ gene-related disease, the likelihood of detecting the kif1Bβ gene-related disease at an early stage is increased by setting up a frequent preventive health plan can do. Also, if it is post-onset, a reasonable prediction of treatment or choice is possible through the classification or differentiation of the kif1Bβ gene-related disease. The test of the present invention is also useful for determining whether the drug obtained by the screening of the present invention is effective. These tests allow the realization of tailor-made treatment according to the individual's kif1Bβ gene-related disease.
[0050]
In the above-described test of the present invention, a polynucleotide containing a part of the nucleotide sequence of the kif1Bβ gene or a sequence complementary thereto, or an antibody against the KIF1Bβ protein is used. The polynucleotide comprising a portion of the kif1Bβ gene nucleotide sequence or a complement thereof may be a polynucleotide comprising the nucleotide sequence of the gene or a portion of the complement thereof in mRNA, cDNA, or genomic DNA; and For example, sequences in protein coding regions, 5 'and 3' UTS, introns, exons, and sequences in transcriptional regulatory regions. The above-mentioned polynucleotide is preferably a polynucleotide comprising contiguous nucleotides in the kiflB gene nucleotide sequence or its complement, which is at least 15 nucleotides or more, preferably 16 nucleotides or more, and more preferably 17, 18 or more nucleotides. These polynucleotides may be DNA or RNA. It may also be a polynucleotide comprising a modified nucleotide. Polynucleotides may be used in tests, as probes, as polynucleotides immobilized in DNA microarrays, or as primers for RT-PCR. Antibodies are also useful for detecting the expression of the kif1Bβ gene at the protein level, and for performing tests through protein structural analysis. The present invention provides a test or diagnostic reagent for a kif1Bβ gene-related disease, comprising the polynucleotide or the antibody. Polynucleotides and antibodies may be bound to a carrier or support. For example, it may be in the form of a DNA microarray or the like. In addition, polynucleotides and antibodies may be suitably combined with solvents and solutes. The test and diagnostic reagents of the present invention are provided, for example, as aqueous solutions. The reagents of the present invention may be test and diagnostic kits, which are placed in suitable containers (including packaging and / or instructions). The test and diagnostic reagents of the present invention are preferably in their containers, packaging, or instructions, which can be used for testing and diagnosing kif1Bβ gene-related diseases, including Charcot-Marie-Tooth disease type 2A. Information, or information indicated in other printed materials, URLs, etc. is attached to this reagent.
[0051]
The present invention also relates to a method of screening for a compound that binds to the polypeptide of the present invention. The screening method of the present invention includes the following. (A) contacting the test compound with the polypeptide of the present invention, (b) detecting the binding activity between the polypeptide and the test compound, and (c) binding to the polypeptide of the present invention. Step of selecting a compound.
[0052]
The polypeptide that binds to the polypeptide of the present invention may be, for example, a cell culture supernatant or extract (which is expected to express a polypeptide that binds to the polypeptide of the present invention), and an affinity column (this (To which the polypeptide of the present invention is attached (immobilized)), and the polypeptide that specifically binds to this column can be purified.
[0053]
Alternatively, polypeptides that bind to a polypeptide of the invention can be screened by Western blot or a two-hybrid system. In the former method, a cDNA library using a phage vector is prepared from a tissue or cell (eg, brain, neuron, etc.) that is expected to express a polypeptide that binds to the polypeptide of the present invention, and is then prepared on agarose. The polypeptide is expressed from the cloned cDNA, transferred to a filter, fixed and reacted with the labeled polypeptide of the invention to detect plaques expressing the bound polypeptide. In the latter method, the polypeptide of the invention is expressed as a fusion protein with a test protein, such as a GAL4 DNA binding region, and a GAL4 transcriptional activation region, and the binding of the polypeptide of the invention to the test protein is GAL4 DNA It is detected by the expression of a reporter gene linked downstream of the promoter having the binding sequence of the binding protein.
[0054]
Other screening methods include: (1) contacting a synthetic compound, natural protein, or random phage peptide display library with an immobilized polypeptide of the invention, and detecting binding molecules. And (2) isolating compounds that bind to the polypeptide of the present invention using high-throughput based on combinatorial chemical technology.
[0055]
Test samples used for screening include, but are not limited to, cell extracts, expression products of gene libraries, synthetic low molecular weight compounds, synthetic peptides, natural compounds, and the like. Those test samples used for screening may be labeled prior to use as an opportunity request. Labels include, but are not limited to, for example, radioactive labels and fluorescent labels.
[0056]
Furthermore, the present invention also relates to a method for screening a candidate compound for treating, alleviating or preventing a kif1Bβ gene-related disease using the transgenic non-human vertebrate of the present invention. For example, in a transgenic non-human vertebrate, the expression of the polypeptide of the present invention is enhanced by substituting, deleting, adding, and / or inserting at least a portion of an endogenous polynucleotide encoding the polypeptide of the present invention. Suppressed vertebrates can be used for the screening methods of the invention. This is because the vertebrate exhibits symptoms of a kif1Bβ gene-related disease such as Charcot-Marie-Tooth disease type 2A. The screening method of the present invention includes the following steps.
(A) administering a candidate compound to the transgenic non-human vertebrate of the present invention;
(B) observing changes in the symptoms of the kif1Bβ gene-related disease in the transgenic non-human vertebrate; and
(C) a step of selecting a candidate compound that alleviates the symptoms of the kif1Bβ gene-related disease.
[0057]
For example, candidate compounds used in the screening of the present invention include natural or synthetic compounds, various organic compounds, natural or synthetic polysaccharides, proteins, peptides, products of gene libraries, cell extracts, and bacteria or fungi. The following components can be mentioned. These candidate compounds may be administered orally or parenterally to transgenic non-human vertebrates for a kiflBβ gene-related disorder.
[0058]
When the polypeptide of the present invention or the compound isolated by the screening method of the present invention is used as a drug, they can be directly administered to a patient or can be administered after being formulated by a known pharmaceutical method. . For example, it can be formulated and administered in an appropriate combination with a pharmacologically acceptable carrier or medium (sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, etc.). Administration to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, or orally by a method known to those skilled in the art. The dosage varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dosage. If the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art.
[0059]
Any patients, patient applications, publications cited in the present invention are incorporated by reference.
[0060]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is described in detail below with reference to examples, but should not be construed as limited thereto.
[0061]
Example 1 A new motor, KIF1Bβ, associates with synaptic vesicle precursors.
Molecular cloning, immunoblot, immunohistochemistry, subcellular fractionation, GST pulldown, and vesicle immunoprecipitation were performed as previously described (Setou, M et al. (2000) Science 288 (5472), 1796- 802). Immuno-EM of vesicles from the cauda equina of rats was performed according to the method described previously (Takeda, S et al. (2000) J. Cell Biol. 148 (6), 1255-65). Anti-synaptotagmin monoclonal antibody 1D12C5 was donated by Dr. Takahashi Masami (Mitsubishi Kasei Life Science Institute, Japan). Anti-synaptophysin and anti-SV2 monoclonal antibodies were a gift from Dr. A. Davies and Dr. I. Jones (Inst Viol & Enviol Microbiol). Anti-SNAP-25 and anti-Mint1 (mLin-10); anti-staxin; anti-COX I; anti-GFP antibodies were purchased from BD Transduction Laboratories; Sigma; Molecular Probes; and Clontech, respectively.
[0062]
Mouse kif1Bβ cDNA was cloned from a 4-week-old ICR mouse brain cDNA library probed with a cDNA fragment of the kif1B motor domain (Nangaku, M et al. (1994) Cell 79 (7), 1209-20). Sequencing of this cDNA revealed that this new and long isoform consisted of 1770 amino acids with a predicted molecular weight of 199 kD (GenBank accession number AB023656; Nakagawa, T et al. (1997) Proc Natl. Acad. Sci. USA 94 (18) 9654-9). Its N-terminal motor domain (1-660aa) was 100% identical at the nucleotide level to the N-terminal motor domain of KIF1Bα. However, the other domains of the two did not share significant homology, suggesting that alternative splicing machinery generated two different motors from a single gene (FIG. 1A).
[0063]
A rabbit polyclonal antibody specific for the KIF1Bβ tail domain was raised against a synthetic peptide TTTFESAITPSESSGYDSADVESLVDREKELAC (SEQ ID NO: 3) that is not homologous to the KIF1A tail and corresponds to 1505-1536aa. Using this antibody, we characterized the cargo organelle of KIF1Bβ and found a single band of the expected size in the brain, and another band in other tissues (possibly a splicing variant). , Or a post-translational modified form) (FIG. 1B; Conforti, L et al. (1999) Mamm. Genome 10 (6), 617-22; Gong, TW et al. (1999) Gene 239 (1), 117-27). This antibody does not cross-react with KIF1A. This is because the present inventors have selected a KIF1Bβ-specific sequence as an antigen. KIF1Bβ is widely distributed in both the cell body and axons of neurons (FIGS. 1C and D, see also FIG. 8), and its tail domain is based on amino acids of the KIF1A motor of synaptic vesicle precursors. We tested the association of KIF1Bβ with various synaptic proteins because they have 61% amino acid identity (Okada, Y et al. (1995) Cell 81 (5), 769-80). Upon density gradient centrifugation, KIF1Bβ fractionated into the same fraction as membrane organelles containing the synaptic vesicle protein synaptotagmin (FIG. 2A). By GST pull-down and immunoprecipitation assays (using mouse brain membrane vesicle fraction (P3)), KIF1Bβ specifically associates with vesicles containing synaptic vesicle proteins synaptotagmin, synaptophysin, and SV2 It was shown. However, no specific association was detected using the synaptic plasma membrane protein SNAP-25 and syntaxin, the mitochondrial marker COXI, or the KIF17-related protein mLin-10 (FIGS. 2B and C; Setou, M et al). (2000) Science 288 (5472), 1796-802). These results were further confirmed by immunoelectron microscopy (immunoEM), suggesting that KIF1Bβ significantly co-localizes with synaptic vesicle proteins on the vesicle membrane (FIG. 2D).
[0064]
Example 2 kif1B^-/-Lethal neuropathy in mice
To assess the functional significance of KIF1B in vivo, we generated knockout mice by gene targeting. We used the positive selection cassette to replace the 2.2 kb portion of the motor domain containing the coding region of the ATP-binding consensus P loop (FIG. 3). In particular, a mouse kif1B genomic clone was obtained from the λEMBL3 genomic library of ES cell line J1 (Tanaka, Y et al. (1998) Cell 93 (7), 1147-58). A 2.2 kb region spanning between the P-loop exon and part of the upstream exon (P-1) was replaced by a neo-selection cassette without poly (A) and as described previously. A knockout procedure was performed (Harada, A et al. (1994) Nature. 369 (6480), 488-91). Chimeric mice were backcrossed to C57BL / 6J female mice in a special sterile environment and two independent strains were used simultaneously. The wild-type kif1B allele was detected by PCR amplification using the following primer set: 5′-CGCTAGGGTTAAAGCACACGCTAC-3 ′ (SEQ ID NO: 4), and 5′-TGAACTTAGAAATCCACCTGCCTC-3 ′ (SEQ ID NO: 5). The neo transgene was amplified as previously described (Tanaka, Y et al. (1998) Cell 93 (7), 1147-58).
[0065]
kif1B^-/-The pups delivered at a normal Mendelian ratio, but died within 30 minutes of birth. Their alveoli did not dilate, and thus apnea could fully explain the cause of neonatal death (FIG. 3E). This apnea was probably not of peripheral origin. This is because the histology and ultrastructure of the lungs and intercostal muscles involved in respiration did not show any significant defects (see FIG. 9).
[0066]
Newborn kif1B^-/-The pups showed multiple neurological abnormalities. They are insensitive to pinching stimuli (Table 1) and their body postures are drooping forelimbs and extremely lordotic (FIG. 3D), which means that Indicates an overall decline in the function of kif1B^-/-The brain was about 10% smaller in size than the control. In cross-section, the cytoplasm, organization, and development of brainstem nuclei and commissure fibers were significantly affected (FIGS. 3F-I, and also see FIG. 10). The number of neuronal cell bodies was less than 25% of the number in controls. kif1B^-/-Loss of neurons in the respiratory center, or in the dorsal and ventral medulla reticularis (MdD and MdV), may be a major cause of neonatal apnea. Hippocampal cytoplasm and organization were also affected (FIGS. 3H and I)
[0067]
(Table 1) Neonatal growth abnormalities and decreased reactivity

All data are shown as mean ± SD.^*p <0.05,^**p <0.001, t test
[0068]
To examine the structure of the synapse, we quantified the synaptic vesicle density. The dorsal horn of the spinal cord was processed at C4 level for conventional electron microscopy. Sixty regions of the synapse were randomly selected from electron micrographs of 100 nm thick sections and described previously (Yonekawa, Y et al. (1998) J. Cell Biol. 141 (2), 431- 41), quantified for 4 pairs of littermates. As a result, we found a significant decrease in the density of nerve terminal synaptic vesicles (see also FIG. 3J and FIG. 9C).
[0069]
Example 3 KIF1Bβ acts on cells autonomously in neurons.
We then proceed with kif1B^{+ / +}Or kif1B^-/-Hippocampal neurons were cultured to evaluate the neuronal loss phenotype in vitro. Neurons were observed 5 consecutive days after plating and established criteria for neuronal differentiation (Goslin, K, and Banker, G (1991) Rat hippocampal neurons in low-density culture.In Culturing Nerve Cells, G. Banker, and K. Goslin, ed. (Cambridge, MA; MIT Press), 251-82), and quantified by classifying into differentiation stages 0 to 4. As previously described (Goslin, K, and Banker, G (1991) Rat hippocampal neurons in low-density culture.In Culturing Nerve Cells, G. Banker, and K. Goslin ed. (Cambridge, MA; MIT Press) Yonekawa, Y et al. (1998) J. Cell Biol. 141 (2), 431-41), culture procedures and quantitative observations were performed. As shown in FIGS.4A-B, kif1B^-/-Neuronal viability dropped significantly after 2 days. Until the fifth day, each kif1B^-/-And kif1B^{+ / +}5%, 10%, and 90% of the neurons survived (FIG. 4B). When knockout neurons were co-cultured with wild-type glia, we concluded that kiflB acts autonomously on cells in neurons in a contribution to cell viability.
[0070]
To assess which isoforms of KIF1B are likely to be responsible for the neuronal loss phenotype, we designed rescue experiments. Fibroblasts were first infected with an adenovirus vector encoding an EGFP-tagged isoform and expression was confirmed by immunoblot (FIG. 4E). For the preparation of adenovirus constructs, the full-length KIF1Bα and KIF1Bβ cDNAs were tagged at the C-terminus with EGFP (Clontech) and as described previously (Nakata, T et al. (1998) J. Cell Biol. 140 (3), 659-74), and ligated into a pAdex-Wt1 vector having a CAG promoter to produce a recombinant adenovirus. 1.0x10 per 35mm diameter dish^FiveIndividual neurons were plated and infected on the day of plating with the same titer of KIF1Bα or KIF1Bβ adenovirus, and survival was observed 5 days later. Infection efficiency is over 90%, and infected kif1B^{+ / +}50-80% of the neurons survived by day 5. Importantly, infection with KIF1Bβ (but not KIF1Bα expressing virus)^-/-Neuronal viability was significantly rescued (FIGS. 4C-D). Any potential defect in mitochondrial trafficking caused by the deletion of KIF1Bα may be attributed to redundant mitochondrial motors (eg, conventional kinesin KIF5A, B, and C: Tanaka, Y et al. (1998) Cell 93 (7), 1147-58; clearly compensated by Kanai, Y et al. (2000) J. Neurosci. 20 (17), 6374-84). This is because no change in mitochondrial distribution was observed in knockout mice, either in vitro (see FIG. 11) or in vivo (not shown). Thus, KIF1Bβ is probably kif1B^-/-It was the major isoform responsible for neuronal loss in mice.
[0071]
Example 4 kif1B^{+ /-}Specific impairment of axonal transport in mice
We then evaluated the heterozygous peripheral nerve protein content to test whether specific transport of synaptic vesicle precursors is impaired by motor protein haploinsufficiency. 2 months old kif1B for research^{+ /-}The mouse sciatic nerve was selected. Because they did not show an overt phenotype that could develop secondary defects. We have found that kif1B^{+ / +}And kif1B^{+ /-}The levels of several proteins in the peripheral nerves of mice were compared in a steady state (FIG. 5A) and in a cumulative manner with 2 hours of nerve ligation (FIG. 5B). Two hours after ligation of the sciatic nerve, a 1 cm long section is collected proximal or distal to the ligated site and immediately contains RIPA buffer, frozen in liquid nitrogen and supplemented with protease inhibitors Crush in a mortar and perform standard methods (Hirokawa, N et al. (1990) J. Cell Biol. 111 (3), 1027-37; Takeda, S et al. (2000) J. Cell Biol. 148 ( 6), processed for immunoblotting using an AP-conjugated secondary antibody according to 1255-65). kif1B^{+ /-}The level of KIF1Bβ protein in the mouse sciatic nerve is^{+ / +}At half the level in mice, this supported the presence of haploinsufficiency (FIGS. 5A-B). With respect to synaptic vesicle proteins, levels of synaptotagmin and SV2 were about 60% of wild type, whereas levels of synaptic membrane proteins SNAP-25 and syntaxin, mitochondrial protein COX I, or control kinesin KIF5A were heterozygous. Did not change significantly (FIG. 5A). Nerve ligation experiments further showed that the transport of synaptic vesicle proteins was specifically reduced (FIG. 5B). In this experiment, the amount of synaptic vesicle protein accumulated at the proximal site in heterozygous axons was lower than that in wild-type axons, but the amount of synapse accumulated in the proximal portion was lower. Plasma membrane protein levels did not change between heterozygous and wild-type axons. Staining of total protein did not show any obvious differences in the levels of major cytoskeletal proteins (FIG. 5D). Thus, a decrease in synaptic vesicle protein transport was less likely due to a non-specific global impairment of axonal transport. In the spinal cord, where the cell bodies of neurons are located, only KIF1Bβ levels were reduced, but synaptotagmin and SV2 levels were not (FIG. 5C). Taken together, these data suggest that synaptic vesicle protein levels were reduced in nerve axons but not in heterozygous cell bodies, which are normally activated by the KIF1Bβ motor. Reflect the specific impairment of axonal transport.
[0072]
Example 5 kif1B with chronic peripheral neuropathy^{+ /-}mouse
kif1B^{+ /-}Mice gradually became staggered after one year of age. They eventually couldn't support their weight, and occasionally could only move by bouncing (Figure 6A). We suspected chronic neuropathy and tested 2-month-old and 12-month-old mice by the following behavioral and physiological tests.
[0073]
kif1B^{+ /-}Mice exhibited progressive muscle weakness and discoordination in behavioral tests. The present inventors performed a fixed bar test and a rotarod test. In the fixed bar test, the mouse was placed on a horizontal wooden bar 20 mm wide. After three trainings, the retention time of the mice was recorded up to 60 seconds as previously described (Kadotani, H et al. (1996) J. Neurosci. 16 (24), 7859-67). In the rotarod test, the mouse was placed on a rotating rod that accelerated from 4 rpm to 40 rpm in 5 minutes. And its retention time was measured essentially as previously described (Jones, B. J., and Roberts, D. J. (1968) J. Pharm. Pharmacol. 20 (4), 302-4). Trials were performed three times a day for five consecutive days. As a result, we found that 12 months old kif1B^{+ /-}Retention times significantly shorter were detected for mice than for controls (Table 2). In fixed bar test, 12 months old kif1B^{+ /-}The mice, which were shivering, were significantly more prone to falling than the controls (Table 2). In rotarod testing, heterozygous mice also showed significant impairment of performance over five consecutive days of training (FIG. 6B; Table 2). 12 months old kif1B^{+ /-}The grasping power of the mouse is kif1B^{+ / +}Although 20-25% weaker than the grip of the mice (Table 2), the sensory impairment tested using the hot plate test was not significant (data not shown). Grasping strength was measured as previously described (Meyer, O. A. et al. (1979) Neurobehav. Toxicol. 1 (3), 233-6). For the hot plate test, mice were placed on a 56 ° C. heat block and latency to licking the forelimbs and hindlimbs was recorded with a 60 second cut-off latency.
[0074]
(Table 2) Animal behavior analysis

Data are reported as mean ± SE in fixed bar and gripping experiments and as mean ± SD in electrophysiological experiments.^*p <0.01 (Mann-Whitney U test).^**p <0.0005 (t-test).^***p <0.05 (Mann-Whitney U test).
[0075]
To assess the nature of muscle weakness, we analyzed the evoked complex action potentials of the 12-month-old sciatic nerve recorded in plantar muscles after direct stimulation at higher levels in the middle (FIG. 6C). ). Peripheral nerve conduction velocities were determined for the sciatic nerve as previously described (Robertson, A et al. (1993) Acta Neuropathol. (Berl) 86 (2), 163-171) with slight modifications. did. All recordings were performed without knowing the genotype. Under ether anesthesia, look at the sciatic nerve and stimulate directly with a 0.1 ms square wave notch in the sciatic or higher level in the middle, using a percutaneously inserted electrode in the plantar muscle of the hind limb. Muscle action potentials were recorded. Motor nerve conduction velocity (MNCV) was calculated by dividing the conduction time by the distance between the stimulation and recording electrodes. As a result, the amplitude is kif1B^{+ /-}Although significantly reduced in mice, the conduction velocity did not show any significant differences (FIGS. 6C-D; Table 2), suggesting that nerve demyelination was not a factor. In addition, histological analysis of the distal muscle showed no significant changes (data not shown). Thus, a defect in the nerve axon could be attributed to kif1B^{+ /-}It is most likely to contribute significantly to peripheral neuropathy in mice.
[0076]
Example 6 Human KIF1B Mutant Causes CMT2A Neuropathy
Finally, we have shown that CMT2A, an autosomal dominant subtype of Charcot-Marie-Tooth disease type II, is a KIF1B-based neuropathy in humans. We doubted this because it is related to the similarity of the mouse phenotype to human disease and the spacing of CMT2A that KIF1B mapped on the first chromosome. We analyzed a previously well-characterized human pedigree of CMT2A, which has an autosomal dominant genetic pattern (FIG. 7A; Saito, M et al. (1997) Neurology 49 (6 ), 1630-5). High molecular weight DNA was prepared from peripheral blood leukocytes from 12 Japanese individuals in CMT2A family # 694 (Saito, M et al. (1997) Neurology 49 (6), 1630-5). Based on the complete exon-intron structure of the human KIF1B gene (Yang, HW et al. (2001) Oncogene, 20 (36), 5075-83), each of the 47 exons was identified by genomic PCR as described. Amplified (Yang, HW et al. (2001) Oncogene, 20 (36), 5075-83) and subjected to SSCP and / or direct sequence analysis. Longer fragments were separated by SSCP analysis using a low pH buffer system (Kukita, Y et al. (1997) Hum. Mutat. 10 (5), 400-7). PCR products were purified and sequenced directly using an ABI sequencer (Perkin Elmer). Among many single nucleotide polymorphisms (SNPs), we have detected that an abnormal SSCP mobility shift in exon 3 is completely associated with clinical signs. This was detected in all four affected individuals of this pedigree, but not in eight unaffected siblings or in 95 unrelated Japanese controls (FIG. 7B). Using the following primer set, 5'-TGGAAGCAATCAAGTAAGTATAGA-3 '(SEQ ID NO: 6) and 5'-CCCCGCATAATGTTCAAGCC-3' (SEQ ID NO: 7), 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds The exon 3 of the human kif1Bβ ortholog was amplified by a 10-minute extension at 72 ° C. under the conditions of 35 cycles. We then directly sequenced the PCR product and performed a heterozygous A → T point mutation (which was⁹⁸Was converted to L) (FIG. 7C).
[0077]
This Q98L mutation is located in the middle of the P-loop, a consensus ATP binding site, and is predicted to disrupt the function of this mechanochemical enzyme. Therefore, we tested the motor activity of recombinant mouse KIF1Bβ using the Q98L mutation. The Q98L mutation was introduced into full-length mouse kiflBβ cDNA by PCR amplification and confirmed by sequencing. First, using standard methods as previously described (Maeda, K et al. (1992) Mol. Biol. Cell 3 (10), 1181-94), we Microtubule-activated ATP turnover of the full-length protein of Q98L recombinant KIF1Bβ was measured at 3.6 ± 0.5 and 0.0 ± 0.4 nmol / mg / min, respectively. Thus, the ATPase activity of the mutated motor was significantly reduced. We further expressed Q98L or wild-type KIF1Bβ in Vero cells and tested their motility in vivo. Vero cells were transfected with cationic lipids, cultured for 24 hours, and immunostained with anti-KIF1Bβ polyclonal antibody and anti-α-tubulin monoclonal antibody DM1A (Sigma). Due to its motility towards the microtubule anode end, overexpressed wild-type KIF1Bβ protein accumulated around the cells (FIG. 7D, left). In contrast, the Q98L mutant protein remained aggregated in the perinuclear region (FIG. 7D, right). These data suggested that the Q98L point mutation resulted in a functional loss of motor activity.
[0078]
Industrial applicability
The present invention provides KIF1Bβ protein, a novel polypeptide having motor activity for transporting synaptic vesicle precursors. The present invention also provides a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a host cell harboring the vector, a method for preparing the polypeptide, and a method for diagnosing a kif1Bβ gene-related disease. provide. Furthermore, the present invention provides a method of screening for a compound that alleviates the symptoms of a kif1Bβ gene-related disease. The polypeptide of the present invention, a gene encoding the polypeptide, and a compound that alleviates the symptoms of a kif1Bβ gene-related disease are therapeutic or prophylactic agents for kif1Bβ gene-related diseases, such as Charcot-Marie-Tooth disease type 2A. Useful for developing.
[Brief description of the drawings]
[0079]
FIG. 1 shows diagrams and photographs showing the molecular cloning and antibody characterization of mouse KIF1Bβ. (A) Schematic representation of the kif1B cDNA. DNA matrix plots (window size = 30, minimum score% = 65) show splicing variations in the tail domain. (B) Immunoblot of adult mouse tissue probed with anti-KIF1Bβ antibody. (C and D) Immunofluorescence microscopy for KIF1Bβ. (C) Motor neurons in the anterior horn. (D) Cross section of the spinal cord showing the axon tract. Bars are 10 μm.
FIG. 2 is a photograph showing the association between mouse KIF1Bβ and synaptic vesicle precursors. (A) Nycodenz density gradient flotation assay, which shows that KIF1Bβ is present in the vesicle fraction containing synaptotagmin. Arrows indicate floated peaks. (B) GST pull-down assay of KIF1Bβ-associated vesicles. Synaptic vesicle proteins were specifically detected in vesicles associated with the KIF1Bβ tail region (aa885-1700), but truncated constructs lacking the pleckstrin homology (PH) domain (ΔPH; aa885-1528) ) Were not detected in the vesicles. (C) Immunoprecipitation of the vesicle fraction of mouse brain using anti-KIF1Bβ and some anti-synaptic vesicle protein antibodies. KIF17 was used as a control. The synaptic vesicle proteins synaptotagmin, synaptophysin, and SV2 were specifically detected in vesicles co-immunoprecipitated with KIF1Bβ, COXI, and cytochrome oxidase I (a marker of mitochondria). (D) Immuno-EM showing coexistence of KIF1Bβ (10-nm gold) and synaptic vesicle protein (5-nm gold) on axonal vesicles. Bars are 100nm. The percentage of the number of vesicles carrying both signals above the number of all vesicles in the randomly selected area was indicated.^**, P <0.001 (independence test, chi-square).
FIG. 3 shows diagrams and photographs showing targeted disruption of the mouse kif1B gene. (A) Targeting strategy for kif1B knockout. The strategy for genomic Southern blot with HincII is also shown. A + T / pau, AT-rich pausing signal; pBS, pBluescriptII-SK (+); Hc, HincII. (B) Genomic southern blot, where the recombinant allele gives an additional band of 2.1 kb. (C) Immunoblot analysis of 18.5 dpc crude extract from mouse brain using anti-KIF1Nβ and anti-KIF1Bα antibodies, respectively. In both cases, kif1B^-/-No band was detected from the sample. (D) Appearance of newborn littermates. kif1B^-/-Note the sagging forelimbs (arrows) and the lordotic posture (arrowhead type), which are specifically found in the pups. (E) HE-stained lung section. Note that alveoli (asterisks) were only inflated with air in control mice. Bars are 25 μm. (FI) kif1B^-/-Brain defects in mice. (FG) kif1B at 18.5dpc^{+ / +}(F), and kif1B^-/-(G) Comparison of respiratory centers in cross sections of the medulla oblongata of mice. MpD: dorsal medulla plexi, MdV: ventral medulla plexi, VLRt: ventrolateral reticular nucleus. Bars are 500 μm. (HI) kif1B at 18.5dpc^{+ / +}(H), and kif1B^-/-(I) Embryonic cerebral hemisphere preforehead (with incomplete formation of commissure fibers). Arrowhead type is anterior commissure; arrow is internal. Bars are 600 μm. (J) kif1B at 18.5dpc^-/-Reduced density of synaptic vesicles in nerve endings in the anterior horn region of the mouse spinal cord. # 1 to # 4 indicate the four different areas counted.
[Fig. 4] kif1B^-/-FIG. 2 shows diagrams and photographs showing neuronal cell death in primary culture of hippocampus. (A) Time course of primary culture of each genotype. Kif1B with significant delay in differentiation^-/-Beware of severe neuronal death of neurons. Arrows indicate dead cells. Bars are 20 μm. (B) Survival and differentiation curves of plated neurons of each genotype. The relative index of the number of neuron cells was calculated with the initial number of cells per unit area as 100%. (CE) This is a relief experiment. (C) Kif1B on day 5 of infection^-/-A rescued culture of hippocampal neurons. Green fluorescence indicates neurons expressing each adenovirus vector, which expresses the kiflB isoform tagged with EGFP. Note that neurons have reached a highly differentiated stage using only the kif1Bβ construct. Arrows indicate dead cells. Bars are 10 μm. (D) kif1B^-/-Quantification of neuronal rescue efficiency.^*p <0.001. β is kif1Bβ, α is kif1Bα, and N is a non-infected control. (E) Immunoblot of infected fibroblast lysates using anti-GFP antibody. Each EGFP-tagged protein yielded a band of the expected molecular weight.
[Fig. 5] kif1B^{+ /-}1 shows photographs showing reduced levels of synaptic vesicle proteins in sciatic nerve axons. (A) Immunoblot of sciatic nerve extract. Each lane was loaded with 10 μg of protein. kif1B^{+ /-}Protein levels in the nerve are kif1B^{+ / +}Expressed as a percentage of the protein level in the nerve. (B) Immunoblot of pooled organelles in the sciatic nerve 2 hours after ligation. Protein levels from the proximal (Prox) and distal (Dist) regions 1 cm relative to the ligated site were compared. (C) Immunoblot of spinal cord extract prepared in the same manner as (A). (D) Protein staining of extracts from sciatic nerve and spinal cord with Coomassie brilliant blue. Each lane was loaded with 10 μg of total protein.
[Fig. 6] kif1B^{+ /-}Figure 3 shows diagrams and photographs showing chronic peripheral neuropathy in mice. (A) Representative posture of a 1.5-year-old mouse. kif1B^{+ /-}Note that the mice could not support their own weight. (B) Retention time in rotor-rod test of 2 and 12 month old mice in 5 consecutive trials. 12 months old kif1B despite improvement after 5 days of training^{+ /-}Mouse holding time is kif1B^{+ / +}It was still significantly shorter than the retention time of the mice (n = 12, p <0.005, ANOVA test). (CD) Electrophysiological analysis of sciatic nerve of 12 month old mice. (C) Typical trace of evoked action potentials in plantar muscle. (D) Quantification of motor nerve conduction velocity (MNCV) and the amplitude of evoked myoelectric potential. Although the amplitude was reduced, the MNCV did not change significantly. This suggests that the disease is of axonal origin.
FIG. 7 shows a diagram and a photograph showing a mutation in loss of function in the human KIF1B gene derived from a CMT2A patient. (A) Genealogy of the # 694 family. Circles are female, squares are male, hatched are dead individuals, white figures are unaffected, black painted figures are affected. (B) Mobility shift in PCR-SSCP analysis for P-loop exon 3. Arrows indicate specially observed shifted bands in affected individuals. (C) Electropherogram of the sequence in the P-loop region. In samples from affected individuals, A and T are read simultaneously at the mutated codon, resulting in a heterozygous Q98L point mutation. (D) Immunofluorescence of Vero cells transfected with either wild type or Q98L mutant KIF1Bβ construct. Red and green signals indicate KIF1Bβ and tubulin, respectively. Note the difference (arrow) in the accumulated position of the KIF1Bβ protein. Bars are 15 μm.
FIG. 8 shows a photograph showing an immunoblot for the spatiotemporal expression pattern of KIF1Bβ. (A) Wide distribution of KIF1Bβ in the 4-week-old mouse nervous system. (B) Continuous developmental expression of KIF1Bβ in the brain. 20 μg of protein was loaded in each lane.
[Fig. 9] kif1B^-/-Shows photographs showing the central (but not peripheral) origin of the neonatal apnea phenotype in mice. (A) Electron micrographs of type II alveolar epithelial cells in the lungs of neonates of each genotype. Note that secreted lamellar bodies (arrows) are found in both samples. This indicates that the function of the lung epithelium^-/-Suggested to be preserved in mice. Bars are 2 μm. (B) Electron micrographs of neonatal intercostal muscle of each genotype. kif1B^-/-The normal development of muscle fibers in mice precludes that this phenotype is of muscle origin. Bars are 2 μm. (C) Electron micrograph of the dorsal horn region of the spinal cord, where the density of synaptic vesicles at nerve endings is kif1B.^-/-Shows a significant decrease in mice. See text. Bars are 400 nm.
FIG. 10 kif1B^-/-1 shows photographs showing incomplete development of the facial nuclei in mice. (A) HE-stained 7 μm thick paraffin-embedded sections of medulla oblongata at each stage of each genotype. The size of the facial nucleus (circled) is kif1B^{+ / +}Gradually increased in the embryo, but kif1B^-/-It is significantly reduced in the embryo, indicating the functional importance of the kiflB gene in neuronal development and survival in vivo. Bars are 400 μm. (B) Toluidine blue stained 7 μm thick paraffin sections of the 18.5 dpc facial nucleus of each genotype (circled). Note the significant decrease in the number of cell bodies. The weaker the staining of the Nissl bodies, the more the kif1B^-/-Reflects the low viability of neurons. Bar is 80 μm.
FIG. 11 shows photographs showing mitochondrial staining in cultured hippocampal neurons of each genotype probed by Mitotracker (Molecular Probes). Note that this mitochondria was similarly distributed in nerve axons of both phenotypes. Bars are 10 μm.

Claims (21)

A substantially pure polypeptide selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;
(B) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein one or more amino acids are substituted, deleted, inserted, and / or added, and the amino acid sequence of SEQ ID NO: 2 is A polypeptide functionally equivalent to the polypeptide comprising; and (c) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, A polypeptide functionally equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. An isolated polynucleotide encoding the polypeptide of claim 1. A vector comprising the polynucleotide according to claim 2. A host cell carrying the polynucleotide according to claim 2 or the vector according to claim 3. 3. A polynucleotide that is complementary to the polynucleotide of claim 2 or its complement, and comprises at least 15 nucleotides. A method for making a polypeptide according to claim 1, comprising the following steps:
(A) culturing the host cell according to claim 4;
(B) allowing the host cell to express the polypeptide; and (c) collecting the expressed polypeptide. An antibody that binds to the polypeptide of claim 1. An antisense polynucleotide to the polynucleotide according to claim 2. A transgenic non-human vertebrate which carries an exogenous polynucleotide encoding the polypeptide of claim 1 in an expressible manner. A transgenic non-human vertebrate, wherein the expression of an endogenous polynucleotide encoding the polypeptide according to claim 1 is suppressed. 11. The transgenic non-human vertebrate of claim 9 or 10, wherein the transgenic non-human vertebrate is a mouse. A method for diagnosing a kif1Bβ gene-related disease, comprising: (a) mutating a gene located in the same allele as the allele in which the gene encoding the nucleotide sequence of SEQ ID NO: 1 is located; Detecting whether or not has;
And wherein the subject is suspected of having a kif1Bβ gene-related disease if the subject has the mutation. A method for diagnosing a kif1Bβ gene-related disease, comprising: (a) detecting the expression level of a gene in a subject, wherein the gene encodes a nucleotide sequence of SEQ ID NO: 1. Is located in the same as the allele in which is located;
And a method in which the subject is presumed to have a kif1Bβ gene-related disease when abnormal expression is detected. 14. The method according to claim 12 or 13, wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A. A method for screening for a compound that binds to a polypeptide according to claim 1, wherein the method comprises:
(A) contacting the test compound with the polypeptide of claim 1;
(B) detecting the binding activity between the polypeptide and the test compound; and (c) selecting a compound that binds to the polypeptide. A method of screening for a candidate compound for treating, ameliorating, or preventing a kif1Bβ gene-related disease, comprising:
(A) administering a candidate compound to the transgenic non-human vertebrate according to any one of claims 9 to 11;
(B) observing changes in the symptoms of the kif1Bβ gene-related disease in the transgenic non-human vertebrate; and (c) selecting a candidate compound that alleviates the symptoms of the kif1Bβ gene-related disease,
A method comprising: 17. The method according to claim 16, wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A. A composition for treating, alleviating, or preventing a kif1Bβ gene-related disease, wherein the composition is a pharmaceutically acceptable compound selected by the method of any one of claims 15 to 17 as an active ingredient. A composition comprising an effective amount and a pharmaceutically acceptable carrier. 19. The composition according to claim 18, wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A. A method for treating, ameliorating, or preventing a kif1Bβ gene-related disease, comprising administering a pharmaceutically effective amount of a compound selected by the method of any one of claims 15 to 17. A method comprising the step of: 21. The method according to claim 20, wherein the kif1Bβ gene-related disease is Charcot-Marie-Tooth disease type 2A.

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