JP4227778B2 - Annexin 5 physiological activity inhibitor - Google Patents

Annexin 5 physiological activity inhibitor Download PDF

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JP4227778B2
JP4227778B2 JP2002243750A JP2002243750A JP4227778B2 JP 4227778 B2 JP4227778 B2 JP 4227778B2 JP 2002243750 A JP2002243750 A JP 2002243750A JP 2002243750 A JP2002243750 A JP 2002243750A JP 4227778 B2 JP4227778 B2 JP 4227778B2
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光盛 汾陽
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Description

【０００１】
【発明の属する技術分野】
本発明は、アネキシン５の生理活性抑制物質及び該生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤、特にアネキシン５の生理活性を抑制するポリペプチド、該ポリペプチドのポリペプチド誘導体又は修飾ポリペプチド、該ポリペプチドをコードするＤＮＡ、該ポリペプチドに特異的に結合する抗体及びそれらの利用に関する。
【０００２】
【従来の技術】
動物個体において、細胞間の情報連絡とそれに基づく細胞の機能変化は、動物個体の恒常性を維持するための基本的プロセスであり、そのために、無機イオンからタンパク質までのさまざまな物質が情報担体として関与している。中でも、カルシウムイオンは、多くの細胞で情報伝達に利用される重要な情報担体である。カルシウムイオンは、各種タンパク質に結合することによって、そのタンパク質機能の賦活化や分布の変化をもたらし、細胞固有の生理機能の発現に寄与している。
【０００３】
近年、新しいカルシウム結合タンパク質として、アネキシンファミリーのタンパク質が注目を集めている（Cell 55, 1-3, 1988、Biochim. Biophys. Acta 1197, 63-93, 1994）。アネキシンは、分子内に約７０アミノ酸残基からなる相同性の高い４回（アネキシン６は８回）の繰り返し構造を持ち、この部位がカルシウムとリン脂質への結合能を示す（Cell 55, 1-3, 1988、Biochemistry 26, 8067-8092, 1987）。カルモジュリンやトロポニンなどの、いわゆるＥＦバンドスーパーファミリーに属するタンパク質とは、カルシウム結合様式の異なる新しいカテゴリーのタンパク質である。アネキシンは、カルシウム濃度に依存したリン脂質結合能という機能を持つこと、動物種間で相同性が極めて高いことから、重要な生理機能を担ったタンパク質であると推定されている。
【０００４】
アネキシンを用いた生化学的実験では、（１）プロスタグランジン産生の律速酵素である、ホスホリパーゼＡ2の抑制作用（Nature 320, 77-81, 1986、J. Biol. Chem. 263, 10, 799-811, 1988）、（２）血液凝固抑制作用（Biochemistry 26, 8087-8092, 1987、Biochemistry 27, 6645-6653, 1988、J. Biol. Chem. 265, 17, 420-423, 1990）、（３）電位依存性のカルシウムチャネル活性、（４）プロテインキナーゼＣの抑制作用（Eur. J. Biochem. 191, 421-429, 1990、Biochemistry 31, 1886-1891, 1992）、（５）膜の融合活性などの細胞内外を作用点とする機能（Cell 55, 1-3, 1988、J. Biol. Chem. 110, 13-25, 1990、J. Cell. Biol. 114, 1135-1147, 1991）が示唆されているものの、作用点を含め実際の生理機能は不明のままであった。
【０００５】
アネキシン５は、各種組織にもっとも豊富に存在するアネキシンのひとつである。上記の機能に加え、アネキシン５はタンパク質キナーゼＣ活性及び血液凝固を阻害する（J. Biol. Chem. 265, 17420-17423, 1990、Biochemistry 31, 1886-1891, 1992）。コラーゲンに対する高い親和性が報告されており、基底膜への細胞付着における役割が示唆されている（FEBS Lett 310, 143-147, 1992）。アネキシン５は、広く組織間に分布しており、その分布は、細胞種に特異的である（J. Histochem. 39, 1189-1198, 1991、Endocrine 5, 9-14, 1996）。また、アネキシン５は卵巣腫瘍のマーカータンパク質として利用可能であり（Gynecol Obstet Invest 32, 107-111, 1991）、アネキシン５の自己抗体は何人かの紅斑性狼瘡（ＬＥ：lupus erythematosus）患者で検出されている（Am. J. Hematol. 47, 56-58, 1994）。こうした観察結果は、アネキシン５が、いくつかの重要な細胞機能に関与していることを示唆している。
【０００６】
本発明者は、アネキシン５が下垂体前葉で発現することを報告した（Biochem. Biophys. Res. Commun. 186, 894-898, 1992）。以前、本発明者の研究室にて行った、抗アネキシン５又は抗ＬＨβ（ＬＨ＝黄体形成ホルモン）のいずれかで染色した下垂体組織隣接切片の免疫組織化学的研究では、性腺刺激ホルモン産生細胞上でアネキシン５が集約的に発現していた（Cell Tissue Res. 292, 85-89, 1998）。しかし、性腺刺激ホルモン産生細胞それ自体がアネキシン５を合成しているのかはわからなかった。というのは、たとえ性腺刺激ホルモン産生細胞と比較すれば弱いものであるにしろ、下垂体細胞の大半がアネキシン５に対する免疫応答性をもつことが判明しているからである（Cell Tissue Res. 292, 85-89, 1998、Endocr. J. 2, 357-362, 1994）。
【０００７】
下垂体前葉におけるアネキシン５のｍＲＮＡの発現は、卵巣切除後に増大し、この増大はエストロゲンの投与によって抑制された（Mol. Cell Endocrnol. 141, 73-78, 1998）。これらの知見から、本発明者は、性腺刺激ホルモン分泌にアネキシン５が果たす役割を提案し、下垂体性腺刺激ホルモン産生細胞におけるアネキシン５の役割について調査した結果、性腺刺激ホルモン放出ホルモン（ＧｎＲＨ）の刺激作用のもとで性腺刺激ホルモン産生細胞によってアネキシン５のｍＲＮＡが発現することを実証し、アネキシン５がインビトロでのＬＨ及び卵胞刺激ホルモン（ＦＳＨ）の放出を刺激することを報告した（Neuroendocrinology 75, 2-11, 2002）。アネキシン５の遺伝子及びアミノ酸配列については、既に報告があり（J. Bio. Chem. 263, 22, 10799-10811, 1988、Gene 149, 2, 253-260, 1994）、それらの配列については、GenBankのデーターベースにおいて、アクセッションナンバー、Ｍ２１７３１によって、ＮＣＢＩのデーターベースにおいて、アクセッションナンバー、ＮＭ００１１５４によって検索することができる。
【０００８】
【発明が解決しようとする課題】
本発明の課題は、アネキシン５の生理活性抑制物質及び該生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤、特にアネキシン５の生理活性を抑制するポリペプチド、該ポリペプチドのポリペプチド誘導体又は修飾ポリペプチドを提供すること、及び該ポリペプチドをコードするＤＮＡ、該ポリペプチドに特異的に結合する抗体及びそれらの利用方法を提供することにある。
【０００９】
【課題を解決するための手段】
本発明者は、アネキシン５の生理的機能及び構造について研究の結果、アネキシン５のアミノ末端側、１５アミノ酸残基からなるアネキシン５作用ペプチド配列があることを見い出し、本発明を完成するに至った。この配列は、アネキシン５に特異的な配列であり、この配列を含有するポリペプチド誘導体又は修飾ポリペプチドを構築することにより、アネキシン５の作用を拮抗的に阻害し、アネキシン５の生理活性を抑制することができる。
【００１０】
例えば、前記するように、アネキシン５については、その生理機能研究が進展し、近年本発明者らによって、このタンパク質が下垂体前葉で、性腺刺激ホルモン（黄体形成ホルモンと卵胞刺激ホルモン）の分泌を著しく刺激することが見い出され、性腺刺激ホルモン産生細胞における当該ホルモンの合成・放出を調節することが確認されている。この下垂体性腺刺激ホルモン産生細胞におけるアネキシン５の作用に拮抗して、本発明の生理活性抑制物質を投与することにより、アネキシン５の作用を阻害して、その結果として性腺刺激ホルモン放出ホルモン（ＧｎＲＨ）の作用を抑制することができる。
【００１１】
また、本発明においては、本発明のアネキシン５における作用特異配列を利用して、該ポリペプチド配列に特異的に結合する抗体を作製することにより、該抗体を用いて、アネキシン５の検出、測定を行うことができる。また、該アネキシン５における作用特異配列をコードするＤＮＡ配列を用いて、該配列のアンチセンスオリゴヌクレオチドを作製することにより、アネキシン５の遺伝子の発現を抑制したり、またアネキシン５の遺伝子の検出用プローブとして用いることができる。
【００１２】
すなわち本発明は、配列表の配列番号１に記載のアミノ酸配列からなるポリペプチド、配列表の配列番号１に記載のアミノ酸配列からなるペプチド配列を持ち、かつアネキシン５の続きのペプチド配列とは相違する続きのペプチド配列を持つポリペプチド誘導体、又は配列表の配列番号１に記載のアミノ酸配列からなるポリペプチドのアミノ基末端をアセチル基で修飾した修飾ポリペプチドからなるアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤（請求項１）からなる。
【００１３】
また本発明は、配列表の配列番号１に記載のアミノ酸配列からなるポリペプチドの１〜数個のアミノ酸を、欠失、置換したペプチド配列又は配列表の配列番号１に記載のアミノ酸配列からなるポリペプチド中に１〜数個のアミノ酸を付加したペプチド配列を持ち、かつアネキシン５の生理活性抑制作用を有するアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤（請求項２）からなる。
【００１４】
さらに本発明は、アネキシン５の生理活性抑制が、アネキシン５の性腺刺激ホルモンの合成・放出を調節する生理活性の抑制であることを特徴とする請求項１又は２記載のアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤（請求項３）からなる。
【００１５】
【発明の実施の形態】
本発明は、配列表の配列番号１のポリペプチド又は配列表の配列番号１のポリペプチド配列を含むアネキシン５の生理活性抑制物質からなる。該配列番号１のポリペプチド配列は、アネキシン５のアミノ末端側、１５アミノ酸残基に相当する。該配列は、アネキシン５の作用ペプチド配列としての機能を有する特異的な配列であり、この配列を含有し、かつアネキシン５の続きの配列とは相違するポリペプチド誘導体又は修飾ポリペプチドを構築することにより、アネキシン５の作用を拮抗的に阻害し、アネキシン５の生理活性を抑制することができる。
【００１６】
本発明のポリペプチド誘導体としては、機能、合成或いは取り扱いの容易性から、配列番号１の１５アミノ酸残基に続いて、数個から数十の適宜のアミノ酸を結合したポリペプチド誘導体を用いることが好ましい。アセチル基等で修飾したポリペプチドとしては、アミノ末端をアセチル基で修飾したポリペプチドが、特に好ましい。
また、本発明は、配列表の配列番号１のポリペプチドの１〜数個のアミノ酸を欠失、置換又は付加したポリペプチド配列を含み、かつアネキシン５の生理活性抑制作用を有する、配列表の配列番号１のポリペプチドの変異体のアネキシン５生理活性抑制物質からなる。
【００１７】
本発明のアネキシン５の生理活性抑制物質はアネキシン５の生理活性抑制剤として使用することができる。本発明のアネキシン５の生理活性抑制物質は、アネキシン５の生理機能を、拮抗阻害的に阻害し、アネキシン５の生理活性を抑制する。例えば、アネキシン５は、視床下部の分泌する性腺刺激ホルモン放出ホルモン（ＧｎＲＨ）の下垂体ゴナドトロフでの作用を媒介することが知られているが、本発明の生理活性抑制物質の投与により、アネキシン５の生理機能を拮抗的に阻害し、性腺刺激ホルモン（ＬＨ及びＦＳＨ）の合成・放出を抑制することができる。
【００１８】
本発明のアネキシン５の生理活性抑制物質を、細胞に投与するには、該生理活性抑制物質を注射剤のような形で投与することができる。この場合には、該生理活性抑制物質を水や生理食塩水又はブドウ糖溶液等に溶解させて調製し、必要に応じて薬学的に許容される緩衝剤、保存剤或いは安定化剤を含有させて投与することができる。本発明のアネキシン５の生理活性抑制物質を、アネキシン５が発現されている卵巣、副腎、心臓、肺、甲状腺などの器官に投与して、アネキシン５が関与している生理機能を抑制することができる。
【００１９】
本発明のアネキシン５の生理活性抑制物質におけるポリペプチドは、周知のポリペプチドの合成法によって合成することができる。また、該ポリペプチドをコードするＤＮＡ配列を用いて遺伝子工学操作によって、製造することもできる。本発明のアネキシン５の生理活性抑制物質である配列表の配列番号１のポリペプチドをコードするＤＮＡ配列は、配列表の配列番号２の配列で示される。該配列は、アネキシン５の特異配列を示す。該ＤＮＡ配列とストリンジェントな条件下でハイブリダイズするアンチセンスオリゴヌクレオチドを構築し、用いることにより、アネキシン５の遺伝子の発現及びアネキシン５の生理活性機能を特異的に抑制することができる。
【００２０】
なお、ここで本発明のアンチセンスオリゴヌクレオチドが「ＤＮＡ配列とストリンジェントな条件下でハイブリダイズする」条件としては、例えば、４２℃でのハイブリダイゼーション、及び１×ＳＳＣ、０．１％のＳＤＳを含む緩衝液による４２℃での洗浄処理を挙げることができ、６５℃でのハイブリダイゼーション、及び０．１×ＳＳＣ、０．１％のＳＤＳを含む緩衝液による６５℃での洗浄処理をより好ましく挙げることができる。ハイブリダイゼーションのストリンジェンシーに影響を与える要素としては、該温度条件以外に種々の要素があるが、当業者であれば種々の要素を組み合わせて、前記ハイブリダイゼーションのストリンジェンシーと同等のストリンジェンシーを実現することができる。
【００２１】
アンチセンスオリゴヌクレオチドを細胞に導入するには、この分野で通常用いられる方法を用いることができる。例えば、アンチセンスオリゴヌクレオチドを、細胞へ直接投与することができる。また、必要に応じて、薬学的に許容される細胞内導入試薬、例えば、リポフェクチン試薬、リポフェクトアミン試薬、ＤＯＴＡＰ試薬等と共に投与することができる。また、該ＤＮＡ配列のアンチセンス鎖の全部又は一部からなる配列を作製して、アネキシン５の遺伝子検出用プローブとして用いることもできる。
【００２２】
更に、本発明においては、本発明のポリペプチドである配列表の配列番号１のポリペプチドを抗原として用いて、抗体を誘導し、該ポリペプチドに特異的に結合する抗体を産生することができる。該抗体は、モノクローナル抗体として、又は、ポリクローナル抗体として、本発明のポリペプチドを抗原として、常法により作製することができる。該抗体とアネキシン５の抗原抗体反応を用いて、周知の免疫検査法により、アネキシン５の検出、測定を行うことができる。
【００２３】
【実施例】
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。
（アネキシン５生理活性抑制物質ＡＮ−１４の調製）
ヒトアネキシン５のｃＤＮＡ配列（データベース：GenBank アクセッションナンバー：Ｍ２１７３１（J. Biol. Chem. 263, 22, 10799-10811, 1988）をもとに、アミノ末端側１５アミノ酸残基からなるペプチドの設計・合成を行い、ＡＮ−１４とした。ＡＮ−１４は、ヒトアネキシン５のアミノ末端側１５アミノ酸残基（配列表配列番号１）を有し、該ペプチドのアミノ末端側（アラニン）をアセチル化したものである。すなわち、Ａｃ−ＡＱＶＬＲＧＴＶＴＤＦＰＧＦＤなるペプチド構造を有する。ＡＮ−１４のＭＡＳＳデータ、及びＨＰＬＣデータを、図１及び図２に示す。
【００２４】
（ＡＮ−１４の下垂体初代培養細胞におけるＧｎＲＨのＬＨ放出促進作用の抑制）
成熟したメスのウイスター・イマミチ系ラットの下垂体より細胞を分離し（Endocrinology 107, 1095-1104, 1980）、１ウエルあたり１０5個の細胞を９６ウエルの培養トレーを用いて１０％牛胎児血清を含むＤＭＥＭ培地中で２日間前培養した。実験当日に牛胎児血清を除いた培養液で３時間前培養し、性腺刺激ホルモン放出ホルモン（ＧｎＲＨ：コンセラール）を加えて１時間培養した。実験群には、ＧｎＲＨに加えてＡＮ−１４を１μｇ／ｍｌの濃度で加えた。各群とも、ｎ＝５で行った。１時間後に培養液を回収して時間分解蛍光免疫測定法でラットの黄体形成ホルモン（luteinizing hormone：ＬＨ）の放出量を測定した（図３）。その結果、ＧｎＲＨのみの群（●−●）と比べ、ＧｎＲＨにＡＮ−１４を添加した群（■−■）ではＧｎＲＨ濃度依存的にＬＨの放出が抑制されることが明らかになった。
【００２５】
（下垂体ゴナドトロフ株化細胞であるＬβＴ２細胞の増殖に対するＧｎＲＨの抑制効果に対するＡＮ−１４の拮抗作用）
下垂体の性腺刺激ホルモン産生細胞（ゴナドトロフ）を腫瘍化して樹立した株化細胞であるＬβＴ２細胞（カリフォルニア大学サンディエゴ校のP. Mellon博士より供与）を継代し、コンフルエントの状態に達する前に分離し、直径３５ｍｍのディッシュに２０万個の細胞を播種して１０％の牛胎児血清を含むＤＭＥＭ培地中で培養した。その際、ＬβＴ２細胞をＧｎＲＨ未添加群（対照群）、ＤＭＥＭ培地に１０-7ＭのＧｎＲＨ（コンセラール）のみを添加した群、ＧｎＲＨと１μｇ／ｍｌのＡＮ−１４を添加した群、ＧｎＲＨと細胞増殖に関わるシグナル伝達因子であるＭＡＰＫＫ（ＭＥＫ）の抑制剤であるＰＤ９８０５９を添加した群の計４群に分けてそれぞれ培養を行い、４日目に細胞をトリプシンで剥離して顕微鏡観測下で細胞数を数えた（図４）。その結果、ＧｎＲＨは細胞増殖を抑制し、ＡＮ−１４はその作用に拮抗することが明らかになった。
【００２６】
（ＧｎＲＨのＬＨβサブユニット遺伝子発現に対する促進効果へのＡＮ−１４の拮抗作用の観察）
ＬＨβプロモーター領域（−７９７〜＋５）をルシフェラーゼ遺伝子発現ベクターであるｐＧＬ３（Promega社製）に導入し、ホタル・ルシフェラーゼ遺伝子をレポーターとするＬＨβプロモーターレポーターベクター（−７９７／＋５ ＬＨβＬＵＣ：図５）を作製した。このレポーターベクターをリポフェクション法によりＬβＴ２細胞に導入後、４２時間の培養を行った。次に、ＬＨβプロモーターレポーターベクター導入ＬβＴ２細胞をそれぞれ、Ｇ−群（プロモーターを入れない空のベクターを導入した対照群）、Ｇ群（１０-7ＭのＧｎＲＨ（コンセラール）を添加）、ＡＮ０．１＋Ｇ群（ＧｎＲＨと０．１μｇ／ｍｌのＡＮ−１を添加）、ＡＮ１＋Ｇ群（ＧｎＲＨと１μｇ／ｍｌのＡＮ−１４を添加）、ＡＮ１０＋Ｇ群（ＧｎＲＨと１０μｇ／ｍｌのＡＮ−１４を添加）の計５群（各群とも、ｎ＝５）にわけ、６時間の培養を行った後、常法により細胞を溶解してルシフェラーゼ活性を測定した（図６）。その結果、ＧｎＲＨは著しくＬＨβサブユニット遺伝子発現を促進し、ＡＮ−１４は濃度依存的にＧｎＲＨの作用に対して拮抗することが明らかになった。
【００２７】
【発明の効果】
本発明により、アネキシン５の生理活性抑制物質及び該生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤を提供することによって、アネキシン５の生理活性を調整することができる。例えば、本発明のアネキシン５の生理活性抑制物質を、アネキシン５が発現している卵巣、副腎、心臓、肺、甲状腺などの器官に投与して、アネキシン５が関与している生理機能を抑制することができる。これによって、例えば、性ホルモンの分泌を抑制して、性ホルモン依存性疾患に対する予防或いは治療を可能とする。
また、本発明によって、アネキシン５の特異配列であるポリペプチドをコードするＤＮＡ、そのアンチセンスオリゴヌクレオチド、更には本発明のポリペプチドに特異的に結合する抗体を提供することによって、アネキシン５の遺伝子の発現の調整を行ったり、或いはアネキシン５の検出、測定を可能とする。これらはアネキシン５の生理機能の解明に役立ち、かつアネキシン５の生理活性調節にも役立つものであり、アネキシン５に関する機能障害の予防や治療に役立つものである。
【００２８】
【配列表】
SEQUENCE LISTING
<110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION
<120> Bioactive substances of Annexin 5 (AN-14)
<130> YG2003-24PCT
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 15
<212> PRT
<213> Homo sapiens
<400> 1
Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
<210> 2
<211> 45
<212> DNA
<213> Homo sapiens
<220>
<221> misc#feature
<223> Base Sequence 145-190 of Annexin 5
<400> 2
gca cag gtt ctc aga ggc act gtg act gac ttc cct gga ttt gat 45
Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
【図面の簡単な説明】
【図１】本発明の実施例において調製された、本発明のアネキシン５の生理活性抑制物質ＡＮ−１４のＭＡＳＳスペクトル分析の結果を示す図である。
【図２】本発明の実施例において調製された、本発明のアネキシン５の生理活性抑制物質ＡＮ−１４のＨＰＬＣの結果を示す図である。（紫外線波長：２１０ｎｍ、溶出：Ａ溶液：０．１％ＴＦＡ／Ｈ2Ｏ、Ｂ溶液：０．１％ＴＦＡ／ＡＣＮ、グラジエント：１０％〜６０％のＢ溶液で３０分間、流速：１．０ｍｌ／分）
【図３】ラット下垂体初代培養細胞におけるＧｎＲＨのＬＨ放出量に対するＡＮ−１４の影響を時間分解蛍光免疫測定法により測定した結果を示す図である。
【図４】下垂体ゴナドトロフ株化細胞であるＬβＴ２細胞の増殖に対するＧｎＲＨの抑制効果に対するＡＮ−１４の拮抗作用を示す図である。
【図５】ＬＨβプロモーター領域（−７９７〜＋５）をルシフェラーゼ遺伝子発現ベクターであるｐＧＬ３に導入して作製した、ＬＨβプロモーターレポーターベクターを示す図である。
【図６】ＧｎＲＨのＬＨβサブユニット遺伝子発現に対する促進効果へのＡＮ−１４の拮抗作用をルシフェラーゼ活性を測定することにより解析した結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an annexin 5 physiological activity inhibitor and an annexin 5 physiological activity inhibitor comprising the physiological activity inhibitor as an active ingredient, particularly a polypeptide that inhibits the physiological activity of annexin 5, a polypeptide derivative of the polypeptide, or The present invention relates to a modified polypeptide, a DNA encoding the polypeptide, an antibody that specifically binds to the polypeptide, and uses thereof.
[0002]
[Prior art]
In animal individuals, information communication between cells and changes in cell functions based on them are fundamental processes for maintaining the homeostasis of animal individuals. For this reason, various substances from inorganic ions to proteins are used as information carriers. Is involved. Among them, calcium ion is an important information carrier used for information transmission in many cells. Calcium ions bind to various proteins, thereby activating the protein function and changing the distribution, thereby contributing to the expression of physiological functions unique to cells.
[0003]
In recent years, annexin family proteins have attracted attention as new calcium-binding proteins (Cell 55, 1-3, 1988, Biochim. Biophys. Acta 1197, 63-93, 1994). Annexin has a highly homologous four-fold structure consisting of about 70 amino acid residues in the molecule (Annexin 6 is eight times), and this site shows the ability to bind calcium and phospholipid (Cell 55, 1 -3, 1988, Biochemistry 26, 8067-8092, 1987). Proteins belonging to the so-called EF band superfamily such as calmodulin and troponin are a new category of proteins with different calcium binding modes. Annexin is presumed to be a protein that has an important physiological function because it has a function of phospholipid binding ability depending on the calcium concentration and has extremely high homology among animal species.
[0004]
Biochemical experiments with annexin, (1), the rate-limiting enzyme in prostaglandin production, inhibition of phospholipase A 2 (Nature 320, 77-81, 1986, J. Biol. Chem. 263, 10, 799 -811, 1988), (2) blood coagulation inhibitory action (Biochemistry 26, 8087-8092, 1987, Biochemistry 27, 6645-6653, 1988, J. Biol. Chem. 265, 17, 420-423, 1990), ( 3) Voltage-dependent calcium channel activity, (4) Inhibitory action of protein kinase C (Eur. J. Biochem. 191, 421-429, 1990, Biochemistry 31, 1886-1891, 1992), (5) Membrane fusion Functions such as activity such as inside and outside cells (Cell 55, 1-3, 1988, J. Biol. Chem. 110, 13-25, 1990, J. Cell. Biol. 114, 1135-1147, 1991) Although suggested, the actual physiology, including the point of action, remained unknown.
[0005]
Annexin 5 is one of the most abundant annexins in various tissues. In addition to the above functions, Annexin 5 inhibits protein kinase C activity and blood clotting (J. Biol. Chem. 265, 17420-17423, 1990, Biochemistry 31, 1886-1891, 1992). A high affinity for collagen has been reported, suggesting a role in cell adhesion to the basement membrane (FEBS Lett 310, 143-147, 1992). Annexin 5 is widely distributed between tissues, and its distribution is specific to cell types (J. Histochem. 39, 1189-1198, 1991, Endocrine 5, 9-14, 1996). Annexin 5 is also available as a marker protein for ovarian tumors (Gynecol Obstet Invest 32, 107-111, 1991), and annexin 5 autoantibodies are detected in some lupus erythematosus (LE) patients. (Am. J. Hematol. 47, 56-58, 1994). These observations suggest that annexin 5 is involved in several important cell functions.
[0006]
The inventor has reported that annexin 5 is expressed in the anterior pituitary gland (Biochem. Biophys. Res. Commun. 186, 894-898, 1992). In an immunohistochemical study of an adjacent section of the pituitary tissue stained with either anti-annexin 5 or anti-LHβ (LH = luteinizing hormone) previously performed in the inventor's laboratory, gonadotropin-producing cells Annexin 5 was intensively expressed above (Cell Tissue Res. 292, 85-89, 1998). However, it was not known whether gonadotropin-producing cells themselves synthesize annexin 5. This is because the majority of pituitary cells have been found to be immune responsive to annexin 5, even though they are weaker than gonadotropin producing cells (Cell Tissue Res. 292). , 85-89, 1998, Endocr. J. 2, 357-362, 1994).
[0007]
Annexin 5 mRNA expression in the anterior pituitary gland increased after ovariectomy and this increase was suppressed by estrogen administration (Mol. Cell Endocrnol. 141, 73-78, 1998). Based on these findings, the present inventor proposed the role of annexin 5 in gonadotropin secretion and investigated the role of annexin 5 in pituitary gonadotropin-producing cells. As a result, gonadotropin releasing hormone (GnRH) We have demonstrated that annexin 5 mRNA is expressed by gonadotropin-producing cells under stimulatory effects and reported that annexin 5 stimulates the release of LH and follicle stimulating hormone (FSH) in vitro (Neuroendocrinology 75 , 2-11, 2002). The gene and amino acid sequence of annexin 5 have already been reported (J. Bio. Chem. 263, 22, 10799-10811, 1988, Gene 149, 2, 253-260, 1994). The database can be searched by the accession number NM001154 in the NCBI database by the accession number M21731.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide an annexin 5 physiological activity inhibitor, an annexin 5 physiological activity inhibitor comprising the physiological activity inhibitor as an active ingredient, particularly a polypeptide that inhibits the physiological activity of annexin 5, and a polypeptide of the polypeptide It is to provide a derivative or modified polypeptide, and to provide DNA encoding the polypeptide, an antibody that specifically binds to the polypeptide, and a method for using them.
[0009]
[Means for Solving the Problems]
As a result of research on the physiological function and structure of annexin 5, the present inventor found that there is an annexin 5 acting peptide sequence consisting of 15 amino acid residues on the amino terminal side of annexin 5 and completed the present invention. . This sequence is a sequence specific to annexin 5, and by constructing a polypeptide derivative or modified polypeptide containing this sequence, the action of annexin 5 is antagonistically inhibited and the physiological activity of annexin 5 is suppressed. can do.
[0010]
For example, as described above, the physiological function research of annexin 5 has progressed, and in recent years, the present inventors have secreted gonadotropins (luteinizing hormone and follicle stimulating hormone) in the anterior pituitary gland. It has been found to stimulate significantly and has been confirmed to regulate the synthesis and release of the hormone in gonadotropin producing cells. By antagonizing the action of annexin 5 in this pituitary gonadotropin producing cell and administering the physiological activity inhibitor of the present invention, the action of annexin 5 is inhibited, and as a result, gonadotropin releasing hormone (GnRH) ) Can be suppressed.
[0011]
In the present invention, an antibody that specifically binds to the polypeptide sequence is prepared using the action-specific sequence of Annexin 5 of the present invention, and the annexin 5 is detected and measured using the antibody. It can be performed. In addition, by using the DNA sequence encoding the action-specific sequence in Annexin 5 to produce an antisense oligonucleotide of the sequence, the expression of Annexin 5 gene can be suppressed, or the annexin 5 gene can be detected. It can be used as a probe.
[0012]
That is, the present invention has a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, a peptide sequence consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and is different from the peptide sequence following Annexin 5 polypeptide derivatives having a peptide sequence of continuation or an amino group-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 a physiologically active inhibitor of annexin 5 consisting of modified polypeptide modified with an acetyl group bioactive inhibitors of annexin 5 as an active ingredient (claim 1) or Ranaru.
[0013]
The present invention also includes a peptide sequence obtained by deleting or replacing one to several amino acids of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. An annexin 5 bioactivity inhibitor comprising a polypeptide having a peptide sequence added with one to several amino acids in a polypeptide and having an annexin 5 bioactivity inhibitory substance as an active ingredient (claim) 2) .
[0014]
Further, in the present invention, the suppression of the physiological activity of annexin 5 according to claim 1 or 2 is characterized in that the suppression of the physiological activity of annexin 5 is suppression of the physiological activity that regulates the synthesis and release of gonadotropin of annexin 5 It comprises an annexin 5 bioactivity inhibitor (Claim 3) containing a substance as an active ingredient .
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The present invention comprises an annexin 5 physiological activity inhibitor comprising the polypeptide of SEQ ID NO: 1 in the sequence listing or the polypeptide sequence of SEQ ID NO: 1 in the sequence listing. The polypeptide sequence of SEQ ID NO: 1 corresponds to 15 amino acid residues on the amino terminal side of annexin 5. Constructing a polypeptide derivative or modified polypeptide which is a specific sequence having a function as an action peptide sequence of annexin 5 and which contains this sequence and is different from the sequence of annexin 5 Thus, the action of annexin 5 can be antagonistically inhibited, and the physiological activity of annexin 5 can be suppressed.
[0016]
As the polypeptide derivative of the present invention, a polypeptide derivative in which several to several tens of appropriate amino acids are bonded to the 15 amino acid residues of SEQ ID NO: 1 is used from the viewpoint of function, synthesis, or ease of handling. preferable. As the polypeptide modified with an acetyl group or the like, a polypeptide in which the amino terminus is modified with an acetyl group is particularly preferred.
In addition, the present invention includes a polypeptide sequence in which one to several amino acids of the polypeptide of SEQ ID NO: 1 in the sequence listing are deleted, substituted or added, and has an action of inhibiting the physiological activity of annexin 5, It consists of an annexin 5 physiological activity inhibitor which is a variant of the polypeptide of SEQ ID NO: 1.
[0017]
The annexin 5 physiological activity inhibitor of the present invention can be used as an annexin 5 physiological activity inhibitor. The physiological activity inhibitor of annexin 5 of the present invention inhibits the physiological function of annexin 5 in a competitive and inhibitory manner and suppresses the physiological activity of annexin 5. For example, Annexin 5 is known to mediate the action of the hypothalamic secretory gonadotropin-releasing hormone (GnRH) on the pituitary gonadotroph, but by the administration of the physiologically active inhibitor of the present invention, Annexin 5 Can be antagonistically inhibited, and synthesis and release of gonadotropins (LH and FSH) can be suppressed.
[0018]
In order to administer the physiological activity inhibitor of annexin 5 of the present invention to cells, the physiological activity inhibitor can be administered in the form of an injection. In this case, the physiologically active inhibitory substance is prepared by dissolving in water, physiological saline, glucose solution or the like, and contains a pharmaceutically acceptable buffer, preservative or stabilizer as necessary. Can be administered. It is possible to administer the physiological activity inhibitor of annexin 5 of the present invention to organs such as ovary, adrenal gland, heart, lung, thyroid and the like in which annexin 5 is expressed to suppress physiological functions involving annexin 5. it can.
[0019]
The polypeptide in the physiological activity inhibitor of annexin 5 of the present invention can be synthesized by a known polypeptide synthesis method. It can also be produced by genetic engineering operations using a DNA sequence encoding the polypeptide. The DNA sequence encoding the polypeptide of SEQ ID NO: 1 in the Sequence Listing which is a substance that suppresses the physiological activity of Annexin 5 of the present invention is represented by the sequence of SEQ ID NO: 2 in the Sequence Listing. This sequence shows the specific sequence of annexin 5. By constructing and using an antisense oligonucleotide that hybridizes with the DNA sequence under stringent conditions, the expression of annexin 5 gene and the physiologically active function of annexin 5 can be specifically suppressed.
[0020]
Here, the conditions under which the antisense oligonucleotide of the present invention “hybridizes with a DNA sequence under stringent conditions” include, for example, hybridization at 42 ° C., 1 × SSC, 0.1% SDS Washing treatment at 42 ° C. with a buffer solution containing 35 μg, hybridization at 65 ° C., and washing treatment at 65 ° C. with a buffer solution containing 0.1 × SSC, 0.1% SDS Preferable examples can be given. There are various factors that affect the stringency of hybridization, in addition to the temperature conditions. Those skilled in the art can combine various components to achieve stringency equivalent to the stringency of hybridization. can do.
[0021]
In order to introduce an antisense oligonucleotide into a cell, a method commonly used in this field can be used. For example, antisense oligonucleotides can be administered directly to cells. Moreover, it can administer with a pharmaceutically acceptable intracellular introduction | transduction reagent, for example, a lipofectin reagent, a lipofectamine reagent, a DOTAP reagent etc. as needed. Alternatively, a sequence comprising all or part of the antisense strand of the DNA sequence can be prepared and used as a gene detection probe for annexin 5.
[0022]
Furthermore, in the present invention, an antibody can be induced by using the polypeptide of SEQ ID NO: 1 in the sequence listing which is the polypeptide of the present invention as an antigen, and an antibody that specifically binds to the polypeptide can be produced. . The antibody can be prepared by a conventional method using a monoclonal antibody or a polyclonal antibody, and the polypeptide of the present invention as an antigen. Using the antigen-antibody reaction of the antibody and annexin 5, annexin 5 can be detected and measured by a well-known immunoassay.
[0023]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
(Preparation of Annexin 5 physiological activity inhibitory substance AN-14)
Based on the cDNA sequence of human annexin 5 (database: GenBank accession number: M21731 (J. Biol. Chem. 263, 22, 10799-10811, 1988)) Synthesis was carried out to form AN-14, which has 15 amino acid residues (amino acid sequence side SEQ ID NO: 1) of human annexin 5 and acetylated the amino terminal side (alanine) of the peptide. That is, it has a peptide structure of Ac-AQVLRGTVTDFPGFD, and MASS data and HPLC data of AN-14 are shown in FIGS.
[0024]
(Inhibition of LH release promoting action of GnRH in AN-14 primary pituitary cultured cells)
Cells were isolated from the pituitary gland of adult female Wistar-Immati rats (Endocrinology 107, 1095-1104, 1980). 10 5 fetal serum per well using 10 5 fetal serum using a 96-well culture tray For 2 days in DMEM medium. On the day of the experiment, the cells were precultured for 3 hours in a culture medium excluding fetal bovine serum, and gonadotropin-releasing hormone (GnRH: Conceral) was added and cultured for 1 hour. In the experimental group, AN-14 was added at a concentration of 1 μg / ml in addition to GnRH. For each group, n = 5. One hour later, the culture broth was collected and the amount of rat luteinizing hormone (LH) released was measured by time-resolved fluorescence immunoassay (FIG. 3). As a result, it was clarified that in the group (■-■) in which AN-14 was added to GnRH, the release of LH was suppressed in a GnRH concentration-dependent manner as compared with the group containing only GnRH (●-●).
[0025]
(Antagonism of AN-14 against the inhibitory effect of GnRH on the proliferation of LβT2 cells, a pituitary gonadotroph cell line)
Passage of LβT2 cells (provided by Dr. P. Mellon, University of California, San Diego), established by tumorizing pituitary gonadotropin-producing cells (gonadotroph) and separating them before reaching confluence Then, 200,000 cells were seeded in a dish with a diameter of 35 mm and cultured in DMEM medium containing 10% fetal bovine serum. At that time, LβT2 cells were not added with GnRH (control group), only 10 −7 M GnRH (conceral) was added to DMEM medium, GnRH and 1 μg / ml AN-14 were added, GnRH and cells The cells were divided into a total of 4 groups, to which PD98059, an inhibitor of MAPKK (MEK), a signal transduction factor involved in proliferation, was added. On day 4, the cells were detached with trypsin, and the cells were observed under a microscope. The number was counted (FIG. 4). As a result, it was revealed that GnRH suppresses cell proliferation and AN-14 antagonizes its action.
[0026]
(Observation of AN-14 antagonism of GnRH on promoting effect on LHβ subunit gene expression)
The LHβ promoter region (−797 to +5) is introduced into pGL3 (Promega), which is a luciferase gene expression vector, and an LHβ promoter reporter vector (−797 / + 5 LHβLUC: FIG. 5) using the firefly luciferase gene as a reporter is prepared. did. This reporter vector was introduced into LβT2 cells by the lipofection method and then cultured for 42 hours. Next, LHβ promoter reporter vector-introduced LβT2 cells were respectively added to G-group (control group into which an empty vector without a promoter was introduced), G group (10 −7 M GnRH (consellal) was added), AN0.1 + G Group (GnRH and 0.1 μg / ml AN-1 added), AN1 + G group (GnRH and 1 μg / ml AN-14 added), AN10 + G group (GnRH and 10 μg / ml AN-14 added) Divided into 5 groups (n = 5 for each group), after 6 hours of culture, cells were lysed by a conventional method and luciferase activity was measured (FIG. 6). As a result, it was revealed that GnRH significantly promotes LHβ subunit gene expression, and AN-14 antagonizes the action of GnRH in a concentration-dependent manner.
[0027]
【The invention's effect】
According to the present invention, the physiological activity of annexin 5 can be adjusted by providing an annexin 5 physiological activity inhibitor and an annexin 5 physiological activity inhibitor containing the physiological activity inhibitor as an active ingredient. For example, the physiological activity inhibitor of annexin 5 of the present invention is administered to organs such as ovary, adrenal gland, heart, lung, thyroid and the like where annexin 5 is expressed to suppress physiological functions involving annexin 5. be able to. In this way, for example, the secretion of sex hormones can be suppressed to enable prevention or treatment of sex hormone dependent diseases.
Further, according to the present invention, by providing a DNA encoding a polypeptide that is a specific sequence of annexin 5, an antisense oligonucleotide thereof, and an antibody that specifically binds to the polypeptide of the present invention, Or the annexin 5 can be detected and measured. These are useful for elucidating the physiological function of annexin 5, and also useful for regulating the physiological activity of annexin 5, and are useful for the prevention and treatment of dysfunction related to annexin 5.
[0028]
[Sequence Listing]
SEQUENCE LISTING
<110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION
<120> Bioactive substances of Annexin 5 (AN-14)
<130> YG2003-24PCT
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 15
<212> PRT
<213> Homo sapiens
<400> 1
Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
<210> 2
<211> 45
<212> DNA
<213> Homo sapiens
<220>
<221> misc # feature
<223> Base Sequence 145-190 of Annexin 5
<400> 2
gca cag gtt ctc aga ggc act gtg act gac ttc cct gga ttt gat 45
Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
[Brief description of the drawings]
FIG. 1 is a diagram showing the results of MASS spectrum analysis of an annexin 5 physiological activity inhibitor AN-14 of the present invention prepared in an example of the present invention.
FIG. 2 is a diagram showing the HPLC results of an annexin 5 physiological activity inhibitor AN-14 of the present invention prepared in an example of the present invention. (UV wavelength: 210 nm, elution: A solution: 0.1% TFA / H 2 O, B solution: 0.1% TFA / ACN, gradient: 10% to 60% B solution for 30 minutes, flow rate: 1. 0ml / min)
FIG. 3 is a graph showing the results of measuring the effect of AN-14 on the amount of GnRH LH released in rat pituitary primary cultured cells by a time-resolved fluorescence immunoassay.
FIG. 4 is a graph showing the antagonistic action of AN-14 against the inhibitory effect of GnRH on the proliferation of LβT2 cells, which are pituitary gonadotroph cell lines.
FIG. 5 is a diagram showing an LHβ promoter reporter vector prepared by introducing an LHβ promoter region (−797 to +5) into pGL3, which is a luciferase gene expression vector.
FIG. 6 is a graph showing the results of analyzing the antagonism of AN-14 to the promoting effect of GnRH on LHβ subunit gene expression by measuring luciferase activity.

Claims (3)

配列表の配列番号１に記載のアミノ酸配列からなるポリペプチド、配列表の配列番号１に記載のアミノ酸配列からなるペプチド配列を持ち、かつアネキシン５の続きのペプチド配列とは相違する続きのペプチド配列を持つポリペプチド誘導体、又は配列表の配列番号１に記載のアミノ酸配列からなるポリペプチドのアミノ基末端をアセチル基で修飾した修飾ポリペプチドからなるアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤。A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, a peptide sequence comprising the peptide sequence consisting of the amino acid sequence recited in SEQ ID NO: 1 in the sequence listing, and different from the peptide sequence continuing from annexin 5 An annexin containing, as an active ingredient, a substance having a physiological activity inhibitor of annexin 5 comprising a modified polypeptide in which the amino group end of a polypeptide comprising the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is modified with an acetyl group 5. Bioactivity inhibitor of 5 . 配列表の配列番号１に記載のアミノ酸配列からなるポリペプチドの１〜数個のアミノ酸を、欠失、置換したペプチド配列又は配列表の配列番号１に記載のアミノ酸配列からなるポリペプチド中に１〜数個のアミノ酸を付加したペプチド配列を持ち、かつアネキシン５の生理活性抑制作用を有するアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤。1 to several amino acids of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are deleted or substituted, or 1 in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. An annexin 5 physiological activity inhibitor having as an active ingredient an annexin 5 physiological activity inhibitor having a peptide sequence to which several amino acids are added and having an annexin 5 physiological activity inhibitory action. アネキシン５の生理活性抑制が、アネキシン５の性腺刺激ホルモンの合成・放出を調節する生理活性の抑制であることを特徴とする請求項１又は２記載のアネキシン５の生理活性抑制物質を有効成分とするアネキシン５の生理活性抑制剤。 3. The annexin 5 physiological activity inhibitory substance according to claim 1 or 2 , wherein the annexin 5 physiological activity inhibition is an inhibition of a physiological activity that regulates the synthesis and release of annexin 5 gonadotropin. An annexin 5 physiological activity inhibitor .
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