JP3671245B2 - Inositol polyphosphate binding peptide - Google Patents

Description

【００３６】
実施例２
ＳＤＳ−ＰＡＧＥによる分析
上記の精製工程におけるＩＰ４ＢＰ画分をそれぞれＳＤＳ−ＰＡＧＥ（３．７５−１２．５％のグラディエントゲルによるLaemmli法）に供した。なお、ゲル染色にはＣＢＢを用いた。その結果を図２Ａに示す。図において、レーンｂ：界面活性剤による可溶化画分８０μｇ；レーンｃ：ヘパリンアガロース画分３０μｇ；レーンｄ：セファロースＣＬ−６Ｂ画分１０μｇ；レーンｅ：ＤＥ−５２画分２μｇ；レーンｆ：セファロースＣＬ−６Ｂリクロマト画分２μｇのＳＤＳ−ＰＡＧＥパターンを示す。なお、レーンａは分子量マーカー(×１０³）であり、２００：ミオシン；１１６：β−ガラクトシダーゼ；９２：ホスホリラーゼ；６６：ウシ血清アルブミン；４５：オボアルブミンを示す。
また、図２Ｂに、セファロースＣＬ−６Ｂリクロマトの６３〜８１画分のＳＤＳ−ＰＡＧＥパターンを示す。
上記ＳＤＳ−ＰＡＧＥによるＩＰ４ＢＰの分子量測定値は、１４０Ｋ及び６５Ｋであった（図２Ｃ参照）。また、１４０Ｋ分子は６５Ｋ分子の２量体であることも判明した。
【００３７】
実施例３
［ ³ Ｈ］ＩＰ４結合性の測定
ＩＰ４結合測定は、ＰＥＧ沈殿法によった(Maeda, N. ENBO. J., 9, 61-67, 1990)。クロマト各画分測定は、［³Ｈ］ＩＰ４を各画分５０μｌに９．６ｎＭになるように添加した。そのサンプルは、０℃で１０分間インキュベート、２μｌのウシ−ガンマーグロブリン（５０ｍｇ／ｍｌ）と混合し、５０μｌの３０％ＰＥＧ６０００／５０ｍＭトリス塩酸（ｐＨ８．０）をサンプルに加え、０℃で５分間インキュベートした。次に、１万ｇ遠心上清を除去し、沈殿を５００μｌのSOLVABLEに溶解させた。ラジオアクティビティは、液体シンチレーション試薬として５ｍｌのアクアゾルＩＩ(Aquasol II)を加えて測定した。非特異結合は１０μＭの非ラベルＩＰ４存在下で測定した。
更に、生化学的性状解析のために、別のＩＰ４結合測定法も用いた。１０μｌ［³Ｈ］ＩＰ４（最終濃度４．８ｎＭ）、１０μｌガンマーグロブリン（最終濃度５０μｇ）、１０μｌの０．５Ｍグッド緩衝液、１０μｌのイノシトールリン酸液（５０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ、ｐＨ７．４で希釈）、サンプル及び蒸留水で全１００μｌに調整した。０℃で１０分インキュベートした後、３０％ＰＥＧ溶液を１００μｌ加え、上記のように測定した。
【００３８】
実施例４
ＩＰ４ＢＰペプチドの同定
実施例１で得た精製ＩＰ４ＢＰ（約０．３ｍｇ）をＳＤＳ−ＰＡＧＥ（３．７５−１２．５％のグラディエントゲル）に供し、分離した蛋白をＰＶＤＦ膜にトランスファーして１４０Ｋと６５Ｋ蛋白相当バンドをブロットから切り出し、０．５ｍＭ ＮａＯＨ処理し、microfuge-tube中で還元、アルキル化した。２％アセトニトリルで洗浄後、膜片をリジルエンドペプチダーゼで２４時間、３７℃で処理した。その濃度は、７．５％アセトニトリルと０．０５％ＳＤＳを含む１００ｍＭトリス塩酸（ｐＨ８．５）の３００μｌ中で、ＩＰ４ＢＰが１／５０モル濃度になるように調整した。膜片は１万ｇで５分間遠心して除去し、上清を凍結乾燥後、０．１％ＴＦＡに溶解しＨＰＬＣカラム[Waters 600E、Ｃ８(μBONDASPHERE 300A Waters)、カラム形状０．３９×１５ｃｍ]にアプライした。０−３０％ ２−プロパノール／アセトニトリル（７：３）（含０．１％ＴＦＡ）のグラディエントを４５ｍｌ以上になるように実施した（０．５ｍｌ／分）。ペプチドピークは２１５ｎｍでモニターし回収した（図３Ａ参照）。図に示されるように、１４０Ｋが６５Ｋの２量体であることが確認された。
次いで、１４０Ｋの主ピーク（図３Ａに示した、１−７の７つのピーク）を１００μｌ以下に濃縮し、ガス−フェーズ シークエンサー(Applied Biosystem)に供し、それぞれのアミノ酸配列を決定した。各ペプチドフラグメントのアミノ酸配列をラット由来シナプトタグミンＩＩ配列と比較した（図３Ｂ参照）。その結果、当該アミノ酸配列は、ラット由来シナプトタグミンＩＩ配列と一致した。
【００３９】
実施例５
シナプトタグミンＩＩのＣ２Ａドメイン及びＣ末端領域ペプチドに対する抗体の作製
(1) ラット由来シナプトタグミンＩＩのＣ２Ａドメイン（アミノ酸配列番号：１３９−２６７）とグルタチオン Ｓ−トランスフェラーゼ（ＧＳＴと称する）の融合蛋白を大腸菌で発現させて精製した（Smithらの方法による；Gene 67, 31-40, 1988)。即ち、ラット由来シナプトタグミンＩＩのＣ２ＡドメインをコードするｃＤＮＡを常法に準じてＰＣＲ法により増幅・調製し、ｐＧＥＸ−２Ｔベクターにサブクローニングし、融合蛋白を大腸菌ＪＭ１０９で発現させた。菌体をソニケーションし、トリトン Ｘ−１００で最終濃度が１％になるように処理した。更に、１万ｇ遠心上清をグルタチオン−セファロースＣＬ−６Ｂによるアフィニティカラムに付し、当該融合蛋白を精製した。
精製品は１０％ＳＤＳ−ＰＡＧＥで分析確認後、リン酸緩衝液（生理食塩含）に透析し、ニュージーランドホワイトウサギの背中に皮下注した。なお、蛋白溶液１ｍｇ／ｍｌとフロイントコンプリートアジュバント１ｍｌを混合し免疫した。また、２種間後にブースターした後、常法に準じて抗体を得た。
【００４０】
(2) ラット由来シナプトタグミンＩＩのＣ末端領域ペプチド（アミノ酸配列番号：４０９−４２２）のアミノ末端にシステインを付加し、ウシ血清アルブミンに結合させた。当該アルブミンは、フリーのスルヒドリル基と反応し得るマレイミド基を有するヘテロビファンクショナル−クロスリンカーに結合したものである(sulfo-SMCC-cBSA)。５ｍｇのＣ末端ペプチドと５ｍｇのsulfo-SMCC-cBSAを、１ｍｌの０．１Ｍリン酸緩衝液ｐＨ７．２（０．１Ｍ ＥＤＴＡ、０．１５Ｍ ＮａＣｌを含）中で混合し、室温で３時間撹拌させながら反応させた。未反応物はセファデックスＧ−２５カラムでゲル濾過で除去した（０．０８３Ｍリン酸緩衝液ｐＨ７．２及び０．１５Ｍ ＮａＣｌ条件下）。ペプチド−ｃＢＳＡ複合体を含有するボイド画分を、アジュバントとして水酸化アルミニウムを用い、上記と同様にニュージーランドホワイトウサギに免疫し、常法に準じて抗体を得た。
【００４１】
実施例６
イムノブロット法及び免疫沈降法による分析
(1)イムノブロット法
実施例１における可溶化画分（粗精製蛋白）及びセファロースＣＬ−６Ｂリクロマト画分（精製蛋白）をＳＤＳ−ＰＡＧＥ（３．７５−１２．５％のLaemmli系）に供し、蛋白をＰＶＤＦ膜にトランスファーした後、上記の抗体と反応させた。その結果を図４に示す。同図において、レーンａは可溶化上清５０μｇ；レーンｂはセファロースＣＬ−６Ｂリクロマト画分３μｇであり、アミノブラックで染色したもの（Ａ）、Ｃ２Ａ領域に対する抗体で反応させたもの（Ｂ）、Ｃ末端領域に対する抗体で反応させたもの（Ｃ）、コントロール抗体で反応させたもの（Ｄ）を示す。なお、検出に用いるペルオキシダーゼ活性は、Vectastain ABCキットでＤＡＢ染色を行った。
図に示されるように、ラット由来シナプトタグミンＩＩのＣ末端アミノ酸配列（４０９−４２２）及びＣ２Ａ領域（１３９−２６７）に対する抗体は、１４０Ｋ及び６５Ｋの何れとも反応することが判明した。
【００４２】
(2)免疫沈降法
抗Ｃ２Ａ抗体１０μｌ（抗体濃度はＩｇＧとして、２、５、１０μｇ／１０μｌ・５０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ、ｐＨ７．４、０．１Ｍ ＮａＣｌ。また、当該抗体はAmersham製プロテインＡカラムで精製したもの）を精製ＩＰ４ＢＰ（０．３５μｇ）に添加し、０．１Ｍ ＮａＣｌを含むＨｅｐｅｓ−ＮａＯＨ（ｐＨ７．４）緩衝液中で室温３０分間インキュベートした。次に、同緩衝液で懸濁させたプロテインＡ−セファロースＣＬ−６Ｂ懸濁液３０μｌ（プロテインＡパウダー０．３ｍｇ）を加え、当該チューブを４℃で３０分間ゆるやかに振盪させた。更に、プロテインＡ−セファロースＣＬ−６Ｂ粒子を１万ｇ遠心することにより沈殿させ、上清２５μｌの残存［³Ｈ］ＩＰ４結合活性を前述のＰＥＧ沈降法で測定した。なお、コントロール実験はウサギ免疫前のＩｇＧ画分を用いて同様に実施した。その結果を、図５に示す。同図において、○は抗Ｃ２Ａ抗体を、●はコントロール抗体を示す。また、％阻害（▲）は、コントロールＩｇＧでの活性から抗Ｃ２Ａ抗体での活性を差し引いた残存活性を示す。
図に示されるように、抗Ｃ２Ａ抗体は用量依存的に精製ＩＰ４ＢＰのＩＰ４結合活性を低下させた。
上記(1)及び(2)この結果は、実施例４に示した部分アミノ酸配列の分析結果とあわせて、マウス由来精製ＩＰ４ＢＰは、明きらかにマウス由来シナプトタグミンであることを説得せしめる。
【００４３】
実施例７
精製ＩＰ４ＢＰのＩＰ４結合活性の性状解析
精製ＩＰ４ＢＰのＩＰ４結合におけるｐＨ依存性を図６に示した。結合活性は、４．８ｎＭ［³Ｈ］ＩＰ４と０．３５μｇの精製蛋白を５０ｍＭ Ｍｅｓ−ＮａＯＨ（ｐＨ４．５−６．７；○）、５０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ（ｐＨ６．２−７．６；□）、５０ｍＭ Ｅｐｐｓ−ＮａＯＨ（ｐＨ７．３−８．３；△）緩衝液で検討した。非特異的結合は４．８μＭのラベルＩＰ４存在下で測定した。サンプルは、０℃で１０分間インキュベートし、結合はＰＥＧ沈降法により測定した。その結果、至適ｐＨは５．０であることが判明した。
【００４４】
次に、精製ＩＰ４ＢＰに対する［³Ｈ］ＩＰ４の結合の飽和性について検討した（図７参照）。結合測定には、０．３５μｇの精製蛋白でｐＨ６．２（△）及びｐＨ７．４（○）、あるいは０．１μｇの精製蛋白でｐＨ５．０（□）、４．８ｎＭ ［³Ｈ］ＩＰ４、各種濃度のコールドＩＰ４、及び５０μｇガンマグロブリンを、１００μｌの５０ｍＭ Ｍｅｓ−ＮａＯＨ（ｐＨ５．０）、５０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ（ｐＨ６．２及び７．４）下で使用した。サンプルは、０℃で１０分間インキュベートし、結合はＰＥＧ沈降法により測定した。
その結果、結合のHalf-maximal reductionは、ｐＨ６．２では３０ｎＭ、ｐＨ７．４では４０ｎＭ、ｐＨ５．０では１７０ｎＭであった。また、スカッチャード分析では、シングル結合であることを示し、ｐＨ６．２と７．４ではＫｄ値が３０ｎＭ、ｐＨ５．０では、１６０ｎＭ、Ｂｍａｘは、ｐＨ６．２で２．７ｐｍｏｌ、ｐＨ７．４で２．４ｐｍｏｌ、ｐＨ５．０で３５ｐｍｏｌ／μｇ蛋白であることが判明した。
【００４５】
次に、ＩＰ４結合サイトの特異性を、５種類のイノシトールポリリン酸を加えることによって検討した。精製蛋白０．３５μｇを種々の濃度のＩＰ３（▲）、ＩＰ４（●）、Ｉｎｓ ３，４，５，６−Ｐ４（○）、ＩＰ５（■）及びＩＰ６（□）の存在下で、４．８ｎＭ［³Ｈ］ＩＰ４とインキュベートした。結合測定はＰＥＧ沈降法で実施した。その結果を図８に示す。図に示されるように、ＩＰ５はＩＰ４よりも［³Ｈ］ＩＰ４を強く置換した。Ｉｎｓ ３，４，５，６−Ｐ４とＩＰ６は、１００ｎＭでＩＰ４の結合を半分抑制した。しかし、ＩＰ６の場合には、置換曲線の勾配は比較的鋭く、また１０ｎＭで［³Ｈ］ＩＰ４の結合を増加させた。また、３００ｎＭの時、ＩＰ４と同様に［³Ｈ］ＩＰ４の結合を強く抑制した。このことは、［³Ｈ］ＩＰ４結合におけるＩＰ６のアロステリック様効果を意味する。他のイノシトール誘導体では親和性は低かった。
【００４６】
実施例８
マウスＩＰ４ＢＰの遺伝子クローニング
実施例４で決定した、ＩＰ４ＢＰの一部アミノ酸配列Ｋ−３ペプチド（ＶＰＹＱＥＬＧ；即ち、Val-Pro-Tyr-Gln-Glu-Leu-Gly）及びＫ−２ペプチド（ＩＨＬＭＱ；即ち、Ile-His-Leu-Met-Gln）に相当する５’ＧＴＩＣＣＩＴＡ（ＴＣ）ＣＡ（ＡＧ）ＧＡ（ＡＧ）（ＴＣ）ＴＩＧＧ３’及び５’（ＴＣ）ＴＧＣＡＴＩＡ（ＡＧ）（ＡＧ）ＴＧ（ＡＧＴ）ＡＴ３’をＰＣＲのプライマーとして合成した。全ＲＮＡはChomczynskiとSacchiの方法(Anal. Biochem., 162, 156-159, 1987)によりマウス小脳より調製し、またポリ（Ａ）⁺ＲＮＡはoligotex-dT30（日本ロシュ）により選択した。
【００４７】
ＰＣＲ増幅はＡＭＶ逆転写酵素で３５サイクル行い、９４℃２分間変性し、４８℃でアニーリング、７２℃３分間鎖伸長を行った。ＰＣＲ生成物はアガロースゲル電気泳動とGeneclean II kit(Biol 01)で抽出し、マウス小脳由来ｃＤＮＡライブラリーからの２×１０⁶プラークをスクリーニングするのに用いた。ハイブリダイゼーションは、５０％ホルムアミド、２×ＳＣＣ、０．１％ＳＤＳ、１００μｇ／ｍｌサケ精子ＤＮＡ、³²Ｐ−ラベルプローブ（５×１０⁵ｃｐｍ／ｍｌ）で４２℃１２時間の条件で実施した。ハイブリダイズフィルターは０．１×ＳＣＣと０．１％ＳＤＳで４２℃にて２回洗浄した。５つの陽性クローンを選択し、それらのｃＤＮＡをｐＢｌｕｅｓｃｒｉｐｔ ＫＳ（−）(Stratagene)にクローン化した。さらに、そのクローンを、BcaBEST Dideoxy Sequencing Kit(Takara Shuzo)で配列決定をした。５つの重複するクローンからシングルオープンリーディングフレームを含む全ＤＮＡ配列を決定した。当該塩基配列を配列番号１に示す。また、その配列から予想されるアミノ酸配列を配列番号２に示す。この配列は、前述の部分アミノ酸配列と一致した。配列番号１において、実線を付した部分が既に決定された部分アミノ酸配列を示す。
【００４８】
実施例９
ＩＰ４ＢＰの発現と機能確認
ＩＰ４ＢＰのＩＰ４結合活性を確認するために発現プラスミド（ｐＥＦ−ＩＰ４ＢＰ）を調製し、ＣＯＳ７にトランスフェクトし、形質転換体を培養することにより、発現ＩＰ４ＢＰ／ｓｙｔ ＩＩ（ｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩ）を調製した。
即ち、ｐＥＦ−ＢＯＳ(Nucleic Acids Res., 18, 5322, 1990)をXbaIで消化し、クレノーフラグメントで平滑化し、NotIリンカーを結合させた。NotIリンカーを結合させた全ＩＰ４ＢＰ／ｓｙｔ ＩＩ ｃＤＮＡ（１−１５９０塩基対）を、ｐＥＦ−ＢＯＳのNotIサイトに挿入した。生成したプラスミド（ｐＥＦ−ＩＰ４ＢＰ）は、ヒトＥＦ１−αプロモーターとヒトＧ−ＣＳＦポリアデニル化配列の間に全長のＩＰ４ＢＰ／ｓｙｔ ＩＩｃＤＮＡを有する。
ＤＥＡＥ−デキストラン法を用い、プラスミドｐＥＦ−ＩＰ４ＢＰ及び対照としてのｐＥＦ−ＢＯＳをＣＯＳ７にトランスフェクトした。トランスフェクト後、４８時間培養した。
【００４９】
培養後、細胞をホモゲナイズし、ホモゲネートをＳＤＳ−ＰＡＧＥに供し、抗ＩＰ４ＢＰ抗体でイムノブロットした。その結果を図９Ａに示す。同図において、レーン１はコントロール（ｐＥＦ−ＢＯＳトランスフェクトＣＯＳ７のヘパリン結合画分）、レーン２はｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩ、レーン３はマウス小脳ミクロゾーマル画分を示す。
図に示されるように、イムノリアクティブバンドは、ｐＥＦ−ＩＰ４ＢＰでトランスフェクトした細胞では検出されるが、コントロール細胞では検出されなかった。検出されたバンドは６０Ｋと低分子量であった。この分子量は、Ｎ−グリコシダーゼＦで処理した小脳由来ＩＰ４ＢＰ／ｓｙｔ ＩＩと同一であることから、上記の分子量の相違は糖鎖の違いによることが証明された。
また、コントロール（ｐＥＦ−ＢＯＳトランスフェクトＣＯＳ７のヘパリン結合画分）、ヘパリン-アガロースで部分精製したｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩ及び小脳由来ＩＰ４ＢＰ／ｓｙｔ ＩＩ(各５．５μｇ)の［³Ｈ］ＩＰ４結合を比較した。その結果を図９Ｂに示した。図に示されるように、ｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩのＩＰ４結合活性は小脳由来のものと比べて遜色なかった。
【００５０】
実施例１０
ＩＰ４ＢＰのＩＰ４結合領域同定
ＩＰ４ＢＰのＩＰ４結合領域を同定するために、膜貫通領域を除いたＩＰ４ＢＰとＧＳＴの融合蛋白を９種類作製した。
即ち、ＩＰ４ＢＰ／ｓｙｔ ＩＩの種々のドメインをコードするｃＤＮＡ（図１０Ａ参照）をＰＣＲ法で増殖した。増殖したフラグメントは、ｐＧＥＸ−２Ｔにサブクローニングし、ＤＮＡ配列を確認した。ＩＰ４ＢＰ／ｓｙｔ ＩＩの種々のドメインとＧＳＴ（グルタチオン Ｓ−トランスフェラーゼ）との融合蛋白の調製は文献（Gene 67, 31-40, 1988)記載の方法に準じて行い、大腸菌ＪＭ１０９で発現させた。次いで、生成融合蛋白を、グルタチオン-セファロース ４Ｂ(Pharmacia)クロマトグラフィーで精製し、ＳＤＳ−ＰＡＧＥで分析した。
【００５１】
９種類の融合蛋白において、ＧＳＴに結合させたドメインの違いは図１０Ａのようにマップされる。また、９種類の融合蛋白をＧＳＴ−ａ〜ＧＳＴ−ｉで表し、例えば、ＧＳＴ−ａはＧＳＴとドメインａとの融合蛋白を示す。
これらの融合蛋白のＩＰ４結合性を、前述の方法で試験した。その結果を図１０Ｂに示す。
図に示されるように、ＩＰ４結合活性はＣ２Ｂ領域の中央部に存在することが判明した。そのアミノ酸配列は、アミノ酸番号３１５−３４６のＩＨＬＭＱＮＧＫＲＬＫＫＫＫＴＴＶＫＫＫＴＬＮＰＹＦＮＥＳＦＳＦ、即ち、下記式（２）に相当した。
Ile-His-Leu-Met-Gln-Asn-Gly-Lys-Arg-Leu-Lys-Lys-Lys-Lys-Thr-Thr-Val- Lys-Lys-Lys-Thr-Leu-Asn-Pro-Tyr-Phe-Asn-Glu-Ser-Phe-Ser-Phe- （２）
この領域はリジンリッチであり、マイナスにチャージしたＩＰ４の結合に重要であると推測される。
なお、他のリジンリッチ領域、例えばＣ２Ａ領域或いはカゼインキナーゼＩＩの内在性ポリリジン刺激シグナルは、ＩＰ４結合活性は示さなかった。また、このＩＰ４結合領域はイカシナプトタグミンのＰｅｐ２０ペプチド（ＩＫＫＫＫＴＴＶＫＫＣＴＬＮＰＹＹＮＥＳＦ；即ち、Ile-Lys-Lys-Lys-Lys-Thr-Thr-Val-Lys-Lys-Cys-Thr-Leu-Asn-Pro-Tyr-Tyr-Asn-Glu-Ser-Phe）に相当することも判明した。また、このペプチドを、イカ巨大シナプス前終末に注入すると、神経伝達物質の放出が可逆的に抑制されることが示された。
【００５２】
実施例１１
ＧＳＴ−ＩＰ４ＢＰのＩＰ４結合性質
精製ＧＳＴ−ｆ（ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂ）に対するＩＰ４結合のスカチャード分析を行った。その結果を図１１Ａに示す。この結果から、Ｋｄは１６５ｎＭ、Ｂｍａｘは３．８ｐｍｏｌ／μｇ蛋白であった。このＫｄ値は、小脳由来ＩＰ４ＢＰ（Ｋｄ＝４０ｎＭ）よりも４倍も高い値を示した。当該ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂはアフィニティが低いが、その理由は、ＧＳＴと融合蛋白を形成しているためと推定される。
【００５３】
当該ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂに対する各種イノシトールポリリン酸の結合脳を測定した。その結果を図１１Ｂに示す。図に示されるように、結合能は、Ｉｎｓ−１，２，３，４，５，６−Ｐ６（ＩＰ６）＞Ｉｎｓ−１，３，４，５，６−Ｐ５（ＩＰ５）＞Ｉｎｓ−１，３，４，５−Ｐ４（ＩＰ４）＞Ｉｎｓ−２、４，５−Ｐ３＞Ｉｎｓ−１、４，５−Ｐ３（ＩＰ３）＞Ｉｎｓ−１，４−Ｐ２であった。つまり、Ｉｎｓ−１，２、３，４，５，６−Ｐ６とＩｎｓ−１，３，４，５，６−Ｐ５はＩｎｓ−１，３，４，５−Ｐ４より効果的な競合物質であった。このことから、ＩＰ４ＢＰ／ｓｔｙ ＩＩのＣ２Ｂ領域は、ＩＰ４だけでなくＩＰ５及びＩＰ６にも結合することを示している。
精製小脳由来ＩＰ４ＢＰ／ｓｔｙ ＩＩに対しても、ＩＰ６を除いて同様の結果が得られた。また、ヘパリンはＩＰ４のＩＰ４ＢＰ／ｓｔｙ ＩＩへの結合阻害を示すが、ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂでも同様であった。
【００５４】
実施例１２
ＩＰ４の結合部位についての検討
Ｃ２ドメインはシナプトタグミンＩ、ＰＫＣαやｒａｂｐｈｉｌｉｎ ３Ａにも存在することが知られている(Rose John et al. Gene 74, 465-471, 1988）。そこで、Ｃ２Ｂドメインに対するＩＰ４結合が、ＩＰ４ＢＰ／ｓｔｙ ＩＩにユニークであるかどうかを確認した。即ち、実施例１０に記載した方法に準じて、シナプトタグミンＩ、ｒａｂｐｈｉｌｉｎ ３Ａ（ｒｐｈ）及びＰＫＣαの蛋白とＧＳＴとの融合蛋白を調製し、ＩＰ４結合能を測定した。その結果を図１２に示す。なお、同図において、例えば、ＧＳＴ−ＳＴＩ−Ｃ２Ａは、ＧＳＴとシナプトタグミンＣ２Ａドメインとの融合蛋白を意味する。
図に示されるように、ＩＰ４の結合はシナプトタグミンＩ、ＩＩのＣ２Ｂドメインに特異的であることが判明した。
そこで、種々の生物由来シナプトタグミンＣ２Ｂドメインの前記式（２）に相当するアミノ酸配列を比較した（図１３参照）。そして、ＩＰ４の結合部位は、ＧＫＲ（Ｌ／Ｉ）ＫＫＫＫＴ（Ｔ／Ｓ）（Ｖ／Ｉ）ＫＫ、即ち前記式（１）に存在すると推察された。
即ち、図１３の配列において、Ｃ末端側の配列はよく保存されており、シナプトタグミンＣ２Ａドメインでも見られる配列であるが、シナプトタグミンＣ２ＡドメインはＩＰ４に結合しないこと；ＰＫＣα及びｒａｂｐｈｉｌｉｎ ３ＡのＣ２ドメインもＩＰ４には結合しないが、これらのドメインには上記式（１）のアミノ酸配列が存在しないことなどから、式（１）で示されるペプチドがＩＰ４の結合部位であると判断した。
【００５５】
実施例１３
Ｃ２ドメインのホスホリピッド結合性質
シナプトタグミンＩのＣ２Ａドメインがあれば、Ｃａ²⁺依存性ホスホリピドの結合に十分であるとの報告があるが、その詳細は不明である。シナプトタグミンのＣ２Ｂドメインに対するイノシトールホスフェートとホスホリピド結合の関係を明らかにすべく、ＧＳＴ融合蛋白でホスホリピド結合試験を実施した。
即ち、リポソーム（ホスファチジルセリン／ホスファチジルエタノールアミン（１：１、ｗ／ｗ））とＧＳＴ融合蛋白を、５０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ（ｐＨ７．４）、室温で１５分間インキュベートした。１２００ｇで１０分間遠心後、ペレット（ホスホリピド結合画分）と上清（非結合画分）を分画した。次に、両画分を１０％ＳＤＳ−ＰＡＧＥに供した。その結果を図１４に示す。同図において、レーン１：上清−Ｃａ²⁺、レーン２：ペレット−Ｃａ²⁺、レーン３：上清＋１ｍＭ Ｃａ²⁺、レーン４：ペレット＋Ｃａ²⁺；ａ）ＧＳＴ−ＳＴＩＩ−Ｃ２Ａ、ｂ）ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂ、ｃ）ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂ（３２７番目のＬｙｓをＧｌｎに）、ｄ）ＧＳＴ−ＳＴＩ−Ｃ２Ａ、ｅ）ＧＳＴ−ＳＴＩ−Ｃ２Ｂ、ｆ）ＧＳＴ−Ｃ２Ａ＋Ｃ２Ｂ、ｇ）ＧＳＴ−ｒｐｈ−Ｃ２Ａ、ｈ）ＧＳＴ−ｒｐｈ−Ｃ２Ｂ、ｉ）ＧＳＴを示す。
【００５６】
つまり、ＧＳＴ−ＳＴＩ−Ｃ２ＡとＧＳＴ−ＳＴＩＩ−Ｃ２Ａは、Ｃａ²⁺依存性にリポソーム（１：１（ｗ／ｗ）ホスファチジルセリン：ホスファチジルエタノールアミン）に結合したが、ＧＳＴ−ＳＴＩ−Ｃ２ＢとＧＳＴ−ＳＴＩＩ−Ｃ２ＢはＣａ²⁺非依存性に結合することが判明した。また、ＧＳＴ−ｒａｂｐｈｉｌｉｎ３Ａ（ｒｐｈ）−Ｃ２ＡはＣａ²⁺依存性にホスホリピド結合活性を有するが、ＧＳＴ−ｒｐｈ−Ｃ２ＢはＣａ²⁺存在下でも結合しないことが判明した。すなわち、シナプトタグミンやｒａｂｐｈｉｌｉｎ３ＡのＣ２Ａドメインは、ＰＫＣαのＣ２ドメインにその機能が類似してＣａ²⁺依存性であるが、Ｃ２Ｂドメインは異なることが示唆される。ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂの３２７番目のアミノ酸をリジンからグルタミンに変えるとホスホリピド結合活性は失われるが、ＩＰ４結合活性は５０％維持された。すなわち、ＩＰ４とホスファチジルセリンはＣ２Ｂドメインの似た領域に結合するものの、認識サイトは若干異なることが判明した。
【００５７】
【配列表】

【００５８】

【図面の簡単な説明】
【図１】各クロマトグラフィーによるＩＰ４ＢＰの精製工程を示す図である。
【図２】ＩＰ４ＢＰ精製工程における各画分のＳＤＳ−ＰＡＧＥ分析の結果を示す図（Ａ，Ｂ）である。なお、ＣはＳＤＳ−ＰＡＧＥ分析による分子量の測定を示す図である。
【図３】ＳＤＳ−ＰＡＧＥで分離したＩＰ４ＢＰ（１４０ｋ及び６５ｋ）のリジルエンドペプチダーゼ分解物のＨＰＬＣ分析を示す図（Ａ）、及び分離した画分（１−７）のアミノ酸配列とラットシナプトタグミンＩＩのアミノ酸配列の比較を示す図（Ｂ）である。図Ａにおいて、（ａ）は１４０ｋの分解物、（ｂ）は６５ｋ分解物を示す。
【図４】粗及び精製ＩＰ４ＢＰのイムノブロット分析の結果を示す図である。図において、ａは粗ＩＰ４ＢＰ、ｂは精製ＩＰ４ＢＰを示す。また、Ａはアミノブラックで染色したもの、ＢはシナプトタグミンＣ２Ａ領域に対する抗体で反応させたもの、ＣはシナプトタグミンＣ末端領域に対する抗体で反応させたもの、Ｄはコントロール抗体で反応させたものを示す。
【図５】抗体存在下におけるＩＰ４ＢＰのＩＰ４結合特性を示す図である。図において、○はシナプトタグミンＣ２Ａ領域に対する抗体の存在下、●はコントロール抗体存在下を示す。
【図６】ＩＰ４ＢＰのＩＰ４結合に対するｐＨ依存性を示す図である。
【図７】ＩＰ４ＢＰに対する［³Ｈ］ＩＰ４の結合の飽和性及びスカッチャド分析を示す図である。
【図８】ＩＰ４ＢＰと種々のイノシトールポリリン酸との結合特性を示す図である。図において、▲はＩＰ３、●はＩＰ４、○はＩｎｓ ３，４，５，６−Ｐ４、■はＩＰ５、□はＩＰ６を示す。
【図９】遺伝子組換技術により発現したＩＰ４ＢＰ／ｓｙｔ ＩＩ（ｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩ）のイムノブロット分析の結果（Ａ）、及び［³Ｈ］ＩＰ４結合能（Ｂ）を示す図である。
なお、図Ａにおいて、レーン１はコントロール、レーン２はｐＥＦ−ＩＰ４ＢＰ／ｓｙｔ ＩＩ、レーン３はマウス小脳ミクロゾーマル画分を示す。
【図１０】実施例１０で調製した、ＧＳＴと各種シナプトタグミンドメインとの融合蛋白において、ＧＳＴに結合させたシナプトタグミンドメインの位置を示す図（Ａ）、及び当該融合蛋白のＩＰ４結合能を示す図（Ｂ）である。
【図１１】融合蛋白ＧＳＴ−ＳＴＩＩ−Ｃ２Ｂのスカッチャド分析の結果を示す図（Ａ）、及び種々のイノシトールポリリン酸との結合能を示す図（Ｂ）である。
【図１２】ＧＳＴと種々のＣ２ドメインとの融合蛋白のＩＰ４結合能を示す図である。
【図１３】種々の生物由来シナプトタグミンＣ２Ｂドメインの式（２）相当領域のアミノ酸配列を比較した図である。図において、ＭＩＩはマウスシナプトタグミンＩＩ、ＭＩはマウスシナプトタグミンＩ、ＨＩはヒトシナプトタグミンＩ、ＢＩはウシシナプトタグミンＩ、ＲＡはシビレ エイ シナプトタグミンＡ、ＲＢはシビレエイ シナプトタグミンＢ、ＳＩはイカシナプトタグミンＩ、ＤＩはショウジョウバエ シナプトタグミンＩ、ＣＩは線虫シナプトタグミンＩを示す。
【図１４】ＧＳＴ−各種Ｃ２ドメイン融合蛋白についてのホスホリピッド結合性を示す図である。[0001]
[Industrial application fields]
The present invention relates to a peptide having a binding property to inositol (hereinafter referred to as Ins) polyphosphate. More specifically, the present invention relates to an inositol polyphosphate-binding peptide comprising a peptide having a specific amino acid sequence and use thereof.
[0002]
[Prior art]
Cells have a mechanism that shows an accurate cellular response in response to extracellular information (first messengers) such as hormones, neurotransmitters, and growth factors. Some extracellular information activates receptors (G protein-linked) on cell membranes by physiological action, and then activates phospholipase (PLC), which is an effector, to turn on phosphatidylinositol (PI) turnover. Facilitate.
Ins 1,4,5-trisphosphate (IP3) is one of the substances (second messengers) that transmit information in cells produced by this PI turnover. The IP3 signal acts on the IP3 receptor present in the intracellular calcium storage site (such as the endoplasmic reticulum) to induce the release of calcium, leading to a transient increase in calcium in the cytoplasm, and various calcium-dependent It regulates the functions of proteins and enzymes to induce various cellular responses.
The above-mentioned IP3 receptor has already been isolated from mouse and rat cerebellum, and its abundance has been reported particularly in Purkinje cells.
[0003]
The present inventors have also continued research on the IP3 receptor, and have determined the cloning and primary structure of cDNA in the mouse cerebellum and clarified its action. Furthermore, the function of the IP3-sensitive calcium channel has been repeatedly studied by reconstituting the purified protein on a planar bilayer membrane or liposome.
This IP3 is rapidly metabolized. When one phosphate is eliminated, Ins 1,4-bisphosphate or one phosphate is added to metabolize Ins 1,3,4,5-tetrakisphosphate (IP4). Is done.
InsP3 3-kinase is involved as an enzyme involved in this phosphorylation reaction.
The hypothesis that various metabolites of these phosphoinositide cascades act as second messengers in cell transmission is shown in the following paper, but no direct proof has been made except for IP3 (Nature).341, 197-205, 1989).
In particular, the hypothesis that IP4 is involved in homeostasis through the influx of calcium into cells and the regulation of various intracellular calcium pools has been submitted. Since it has not been found, the details of the function of IP4 are not yet known.
[0004]
In a series of studies, the solubilization and purification of IP4 high-affinity binding protein from porcine cerebellum was reported in 1991, and Theibert et al. Reported that two IP4 binding proteins were isolated from rat cerebellum. (Proc. Natl. Acad. Sci.,88, 3165-3169, 1991). However, in those reports, the physiological function of the IP4 binding protein was completely unknown.
Similarly, Ins 1,2,3,4,5,6-hexakisphosphate (IP6) binding protein has also been reported by two groups, and its partial amino acid sequence is AP2 which is a clathrin assembly protein. Has been revealed. This AP2 is probably considered to be a protein involved in endocytic and recycling pathways of synaptic vesicles (Biochme. Biopys. Res. Common.,187, 158-163, 1992; Proc. Natl. Acad. Sci.,89, 8976-8980, 1992).
[0005]
On the other hand, synaptic transmission is triggered by the release of neurotransmitters from nerve endings. The release of neurotransmitters is thought to be performed by so-called opening release accompanied by fusion of synaptic vesicle membranes and presynaptic membranes, but the molecular mechanism is hardly clarified.
Synaptotagmin is a protein involved in the opening release. The protein was originally identified in 1981 as a synaptic vesicle membrane protein, but is also known as a p65 protein due to its molecular weight (J. Cell Biol.,91, 257-269, 1981). As a result of investigation using a monoclonal antibody, this protein was specifically found only in certain endocrine cells such as a wide range of neurons ranging from the central to the peripheral, adrenal medullary cells and pituitary cells. Furthermore, as a result of cell fractionation and electron microscopic observation, it has been found that this protein is localized in synaptic vesicle membranes and part of cell membranes in nerves.
In 1990, the cDNA of this protein was cloned from a rat cDNA library, and its primary structure was revealed (Nature345, 260-263, 1990). At this time, a hypothesis has been proposed that this protein has a role of binding synaptic vesicles to the presynaptic membrane.
[0006]
The molecular structure of synaptotagmin has one transmembrane portion in the molecule, and the amino group side is exposed inside or outside the synaptic vesicle. A sugar chain is bonded to this part, and the amino acid sequence varies greatly between animal species.
In the cytoplasmic part, there is a double repeat structure that is homologous to the C2 domain that is the activity control site of protein kinase C (PKC). This domain is very well retained among animal species and has the ability to bind Ca / phospholipids. Among these, the C2B domain has not been well-characterized yet, unlike the C2A domain that has been well studied. That is, the Ca / phospholipid binding is known only in the C2A domain of synaptotagmin I. In addition, amino acid sequences that can be phosphorylated by PKC or Ca / calmodulin-dependent kinase II have been found.
The cytoplasmic amino acid sequence is very well conserved during evolution, suggesting the importance of this part in synaptic function (J. Biol. Chem.,266, 263, 1991; Neuron6, 993-1007, 1991; J. Biol. Chem.,266, 13548-13552, 1991). In addition, it has been clarified that there are isoforms encoded by two different genes in rat and three different genes in Shibirei. The distribution of synaptotagmin expression in the brain varies between isoforms, and its physiological meaning is still unclear, but it interacts with the cell membrane proteins neurexin, syntaxin, and N-type calcium channels. It is considered to be a protein involved in the binding and fusion of synaptic vesicles with cell membranes (Neuron6, 993-1007, 1991; J. Biol. Chem.,266, 13548, 1991; Science256, 1820-1823, 1992).
[0007]
[Problems to be solved by the invention]
As described above, inositol polyphosphate plays an important role as a second messenger in cells. The receptor of IP3 has been clarified, and as a result, the action of IP3 has become quite clear. However, no receptor or binding protein has been found for inositol polyphosphates such as IP4, Ins 1,3,4,5,6-pentakisphosphate (IP5) and IP6. Therefore, the action / function of inositol polyphosphate such as IP4 has not been fully elucidated. Therefore, many researchers have searched for proteins that specifically bind to inositol polyphosphate such as IP4. If such a protein is found, it can be used to elucidate the action / function of inositol polyphosphate such as IP4, and is useful as a pharmaceutical, a diagnostic agent, etc., and further screens for calcium inhibitors, neurotransmitters, hormone release regulators, etc. Can be used.
[0008]
From these problems, the present inventors have intensively studied the binding protein of IP4 and found that this protein is identical to the above-mentioned synaptotagmin, and found that IP5 and IP6 also bind to this protein. We found that synaptotagmin, a protein, is a binding protein of various polyinositols.
Furthermore, the present inventors have examined the binding site of IP4 in synaptotagmin. As a result, IP4 binds to the C2B domain of synaptotagmin; more specifically, the binding site is a portion having a specific amino acid sequence in the C2B domain. I found out. It was also found that this binding is specific to synaptotagmins I and II, and PKCα, labfilin and the like, which are proteins having the same domain as the C2B domain described above, do not bind to IP4.
The present invention has been made on the basis of the above findings, and the object of the present invention is a polypeptide capable of binding to inositol polyphosphate such as IP4, IP5, and IP6, a screening method using the peptide, and a drug containing the peptide Is to provide.
[0009]
[Means for Solving the Problems]
  The present invention made to solve the above problems
(a) Peptides having an amino acid sequence represented by the following formula (1)InA peptide having a binding property to inositol tetrakisphosphate, inositol pentakisphosphate or inositol hexakisphosphate (hereinafter referred to as inositol polyphosphate for convenience);
    Gly-Lys-Arg-X₁-Lys-Lys-Lys-Lys-Thr-X₂-X₃-Lys-Lys (1)
  (Where X₁Is Leu or Ile, X₂Is Thr or Ser, X₃Represents Ile or Val)
(b) The above peptide, wherein the peptide is a peptide constituting the C2B domain of synaptotagmin I or II (a) Peptides described;
(c) the above (a) Or (b) A method for screening an inositol polyphosphate-like substance or an inositol polyphosphate antagonist, which comprises using the peptide described in 1);
(d) the above (a) Or (b) Inositol Polyphosphate Inhibitor Containing Peptide Described as an Active Ingredient;
It is.
[0010]
  The peptide of the present invention has a binding property to inositol polyphosphate and has a peptide having the amino acid sequence represented by the formula (1).DoIt becomes. As described above, the present inventors have searched for a binding protein for inositol polyphosphate, and as shown in Examples described later, the binding protein was found to be synaptotagmin. As a result of further studies, it was revealed that the binding site with inositol polyphosphate is in the C2B domain of synaptotagmin, more specifically, the peptide portion represented by the formula (1). That is, a peptide comprising the amino acid sequence of the formula (1)DoCan bind to inositol polyphosphate.
[0011]
  Peptide comprising the amino acid sequence of the above formula (1)DoThe peptide can be prepared by a conventional peptide synthesis method such as a solid phase synthesis method. In addition, it can also be obtained from natural products containing the peptide. Examples of such natural peptides include synaptotagmins I and II of humans, mice, rats, cows, squids, millet, Drosophila, nematodes, etc., and these synaptotagmins Examples of the C2B domain can be exemplified.
  In addition, DNA encoding the above-mentioned target peptide is prepared, incorporated into an appropriate expression vector, and an appropriate host (for example, E. coli, Bacillus subtilis, yeast, filamentous fungus, plant cell, animal cell, etc.) Then, the transformant is cultured, and the culture supernatant (and the supernatant after disrupting the cultured cells) is purified according to a conventional method to obtain the target peptide.
  The peptide of the present invention may contain a sugar chain.
[0012]
The peptide of the present invention has binding properties to inositol polyphosphate. Therefore, by using the peptide of the present invention, it is possible to screen for a substance having physiological activity similar to inositol polyphosphate or a substance that antagonizes inositol polyphosphate. For example, [^ThreeH] and the like, in the presence of inositol polyphosphate, the test substance reacts with the peptide of the present invention (competitive reaction method), and if the amount of labeled inositol polyphosphate bound to the peptide of the present invention decreases, the test substance becomes inositol. It is presumed that the substance has a physiological activity similar to that of polyphosphoric acid. As described above, inositol polyphosphate is presumed to bind to a receptor in the cell and to perform actions such as calcium release, neurotransmitter release, and regulation of hormones and enzymes. Therefore, according to the screening method of the present invention, it is possible to perform screening for intracellular calcium release substances, neurotransmitter release substances, and the like.
[0013]
Moreover, since the peptide of this invention couple | bonds with inositol polyphosphate, it acts as an inhibitor of inositol polyphosphate. The inositol polyphosphate inhibitor of the present invention utilizes such an action and is an inositol polyphosphate inhibitor comprising the peptide of the present invention as an active ingredient. Since inositol polyphosphate is considered to have the above-described action, the inositol polyphosphate inhibitor of the present invention can be used as a calcium inhibitor, a neurotransmitter release inhibitor, a hormone release inhibitor, and the like.
The above-mentioned inhibitors can be administered in various preparation forms (for example, liquids, solids, capsules, etc.), but in general, only the peptide of the present invention, which is an active ingredient, or a conventional carrier and an injection or oral It is used as an agent. The injection can be prepared by a conventional method. For example, the peptide of the present invention is dissolved in an appropriate solvent (for example, sterilized water, buffer solution, physiological saline, etc.) and then filtered and sterilized. And then filling by filling into a sterile container. Oral medicines are formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups, etc. It can be prepared according to the conventional method of chemical conversion.
The content of the peptide of the present invention in the preparation can be appropriately adjusted according to the dosage form, applicable disease and the like.
[0014]
In formulating, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Furthermore, the preparation of the present invention may contain additives necessary for formulation, such as excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like. In the case of a liquid preparation, it is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by re-dissolving and adding distilled water for injection at the time of use.
The inhibitor of the present invention can be administered by an appropriate administration route depending on the preparation form. The dosage is appropriately adjusted according to the patient's symptoms, age, weight, and the like.
[0015]
The peptide of the present invention can also be used as a reagent for measuring inositol polyphosphate by using it as a measurement (quantitative or qualitative) reagent for inositol polyphosphate, an affinity carrier in which the peptide of the present invention is immobilized on a conventional insoluble carrier, and the like. can do.
[0016]
Hereinafter, the present invention will be described in more detail.
Since the phosphoinositide cascade has a very close function to the function of the cerebellum, the present inventors purified and isolated the IP4 binding protein using the cerebellum. That is, since IP3 receptors are abundant in Purkinje cells, and InsP3 3 kinase is also localized in Purkinje cells, it was speculated that IP4 was also produced in these cells.
Further, in the conventional reports, since the binding activity of inositol phosphate is relatively high, it is strongly suggested that polyinositol phosphate is involved in the neurological function of Purkinje cells.
[0017]
Therefore, the present inventors first solubilized with a surfactant. By this operation, the IP3 receptor, inositol polyphosphate phosphatase and kinases that affect the IP4 binding ability are extracted, and these proteins were removed by heparin agarose chromatography. That is, phosphatases and kinases are removed in the effluent or 0.5M fraction, and IP3 receptor is removed in 0.5M NaCl. Differences from the previously reported methods for purifying IP4 binding proteins are probably due to differences in the type of surfactant, differences in the behavior of heparin agarose gels, and differences in ion exchange columns.
At this time, some IP4 activity was found in the effluent, but this was probably due to the polymer of the IP4 binding protein.
[0018]
Next, the remaining impurities were removed by ion exchange chromatography DE-52. At this time, in addition to the target protein, low molecular weight compounds remained and were removed by the second gel filtration. The elution pattern in each chromatography is shown in FIG.
After separation by the final Sepharose CL6B, the target fraction was analyzed by SDS-PAGE, and protein bands with molecular weights of 140 k and 65 k were obtained (see FIG. 2). When CBB staining was performed, the strength of the target IP4 binding activity was very well matched. The second gel filtration showed 140 k in the presence of a surfactant, and this molecular weight protein was considered to be a 65 k protein dimer.
As described above, the present inventors have succeeded in isolating a target IP4 binding protein of 0.45 mg from 150 g of mouse cerebellum.
[0019]
In order to determine the amino acid sequence of the protein thus obtained, the present inventors used the following method. That is, 140 k and 65 k proteins were transferred to a PVDF membrane and hydrolyzed with lysyl endopeptidase in the presence of 0.05% SDS. The remaining peptides were then separated by reverse phase chromatography.
As a result, although the 140 k protein was a minor protein, a large amount of this 140 k-derived peptide was recovered in this separation. Among the 140k-derived peptides thus obtained, amino acid sequences were determined for 7 species and searched using the SWISS PROT database, which revealed complete agreement with the amino acid sequence of rat synaptotagmin II (see FIG. 3).
As mentioned in the prior art, synaptotagmin is thought to be a protein that plays a role in the binding and fusion of presynaptic vesicles (Science259, 780-785, 1993; Neuron6, 665-677, 1991). Native rat synaptotagmin is known to form duplexes and to form high molecular weight complexes with presynaptic proteins.
[0020]
Moreover, according to cDNA analysis, the presence of two types of synaptotagmins (I, II) is known, and RNA blotting analysis has a different distribution (J. Biol. Chem.,266, 13548-13552, 1991). That is, synaptotagmin II exists systematically in the old part in the brain. For example, the spinal cord, brain stem, cerebellum and the like. On the other hand, synaptotagmin I exists in a relatively new region, that is, in the cerebral cortex, hippocampus and the like. In the present invention, only synaptotagmin II was obtained, but as described above, synaptotagmin II shows a greater part in the cerebellum.
[0021]
The present inventors further proved that the protein obtained as described above is rat synaptotagmin II by the following method.
A sample extracted with the surfactant of the present invention, which is an antibody against a peptide corresponding to the C-domain homologous to the c-terminal (409-422) of rat synaptotagmin II and PKC regulatory region synthesized by a conventional method When the samples obtained in the final stage were tested by Western blotting, 140 k and 65 k proteins reacted and crossreacted (see FIG. 4).
In addition, a protein having IP4 binding activity was immunoprecipitated in a dose-dependent manner by the C2A antibody (see FIG. 5).
Based on these facts, the present inventors have found that the purified and purified protein is exactly mouse synaptotagmin II as described above.
[0022]
Next, the present inventors used the following method in order to investigate the characteristics of the purified IP4 binding protein / synaptotagmin.
First, based on a conventional method, the pH dependence on IP4 binding was studied (see FIG. 6). In the presence of 4.8 μM unlabeled IP4, the maximum binding capacity of labeled IP4 is seen around pH 5.0.
[0023]
Replacement of labeled IP4 with unlabeled IP4 in the purified sample was carried out at pH 5.0, 6.2, 7.4, 30 nM at pH 6.2, 40 nM at pH 7.4, pH 5. When 0, it was reduced by about half at 170 nM. As a result of Scatchard analysis, Kd was about 30 nM at pH 6.2 and 7.4, and 160 nM at pH 5.0 (see FIG. 7). Bmax was 2.7 pmol, 2.4 pmol, and 35 pmol / protein at pH 6.2, 7.4, and 5.0, respectively. The Hill coefficient was approximately 1.0 without depending on each pH.
[0024]
Next, the specificity at the IP4 binding site was examined by using various inositol derivatives. The result is shown in FIG. As shown, IP5 displaced the binding of IP4 labeled with high intensity. The strength of this substitution ability was stronger than IP4.
Inositol 3,4,5,6-tetrakisphosphate and inositol hexakisphosphate inhibited IP4 binding by about half at 100 nM.
However, in the case of inositol hexakisphosphate, the slope of the displacement curve was relatively sharp, together increasing the binding of labeled IP4 at 10 nM. In addition, at 300 nM, suppression of strongly labeled IP4 binding was observed as with IP4.
This indicated an allosteric effect. Other inositol derivatives used had a relatively low affinity.
As described above, the present inventors have succeeded in isolating and purifying a high-affinity IP4 binding protein from mouse cerebellum, and confirming and characterizing the binding protein.
[0025]
Next, the present inventors studied what part of the binding protein the IP4 binds to and what the function of the domain is.
First, mouse IP4 binding protein / synaptotagmin (IP4BP / syt II) cDNA was cloned in order to know the IP4 binding domain. Screening was carried out by using cDNA prepared by PCR primers corresponding to the partial amino acid sequences described above from a mouse cerebellar cDNA library.
The sequence of the cDNA containing one translation part was determined by five overlapping clones. The thus obtained mouse IP4BP / syt II nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 2, and as expected, it was the same as the sequence of the IP4 binding protein described above. The initiation codon was also identical to the known Kozak sequence. Compared to rat synaptotagmin II, the four amino acid sequences differed only at the N-terminus. That is, the 12th Ile changed from Asn, the 27th changed from Ala to Val, the 39th changed from Thr to Pro, and the 52nd changed from Asp to Glu. The 3 'untranslated region was identical in rat cDNA and contained a repeating structure of TG dinucleotides.
[0026]
Next, in order to confirm the binding activity of gene expression IP4BP / syt II, the following experiment was performed.
An expression plasmid having the cDNA thus obtained was prepared and transfected into normal COS cells. The homogenate of the cells thus expressed was subjected to normal SDS-PAGE, and then immunoblot analysis was performed with an anti-IP4BP / syt II antibody (see FIG. 9A). As a result, the molecular weight was about 60 k, which was slightly lower than IP4BP / syt II obtained from the cerebellum, but this was attributed to the difference in sugar chains. This expressed protein was then solubilized and partially purified on a heparin agarose column. When the purified IP4BP / syt II was examined for IP4 activity by the method described above, it was the same as the cerebellum-derived IP4BP / syt II (see FIG. 9B).
[0027]
Next, in order to analyze the IP4 binding domain of IP4BP / syt II, a fusion protein of IP4BP / syt II and GST excluding the transmembrane region was expressed. As a result of mapping the IP4 binding ability, the amino acid number 315-346 of the C2B domain (IHLMQNGKRRLKKKKTTTVKKKTNLNPYFNESFSF; ie, Ile-His-Leu-Met-Gln-Asn-Gly-Lys-Arg-Leu-Lys-Lys-Lys-Lys-Lys-Lys Thr-Val-Lys-Lys-Lys-Thr-Leu-Asn-Pro-Tyr-Phe-Asn-Glu-Ser-Phe-Ser-Phe-) was found to be important for binding (see FIG. 10). . Moreover, the region was found to be rich in lysine. Surprisingly, this binding region was found to be very similar to the Pep2 peptide of squid synaptotagmin (Nature363, 163-165, 1993).
Furthermore, as a result of detailed examination of the amino acid sequence of the C2A domain of synaptotagmin, the C2 domain of PKCα and rabphilin 3A, and the binding property between these domains and IP4, the binding site of IP4 is GKRLKKKKTTVKK (ie, Gly- Lys-Arg-Leu-Lys-Lys-Lys-Lys-Thr-Thr-Val-Lys-Lys-) site was revealed. As a result of examining the synaptotagmin C2B domain of animals other than mice, it was found that the peptide moiety represented by the formula (1) is essential for binding to IP4.
[0028]
Further, when a Scatchard analysis of IP4 binding to purified GST-f was performed, Kd was 165 nM and Bmax was 3.8 pmol / μg protein (see FIG. 11A). This Kd value was also found to be higher than purified cerebellar derived IP4BP / syt II. In addition, the low affinity for IP4 is expected due to the fusion with GST.
When the IP4 binding ability to the GST-syt II-C2B is compared, Ins 1,2,3,4,5,6-P6 (IP6)> Ins 1,3,4,5,6-P5 (IP5)> Ins 1,3,4,5-P4 (IP4)> Ins 2,4,5-P3> Ins 1,4,5-P3 (IP3)> Ins 1,4-P2 (see FIG. 11B). That is, IP6 and IP5 were competitive with IP4. From these facts, it was found that the C2B domain of IP4BP / syt II binds not only to IP4 but also to IP6 and IP5.
[0029]
Similar results were obtained for purified cerebellum-derived IP4BP / syt II except for IP6. Heparin also showed inhibition of binding of IP4 to IP4BP / syt II, but the same was true for GST-syt II-C2B.
The C2 domain is also known to be present in synaptotagmin I, PKCα and labfilin 3A (Mol. Cell Biol.,13, 2061-2068). Therefore, when it was confirmed whether the IP4 binding to the C2B domain is unique to IP4BP / syt II, it was found that the binding of IP4 is specific to the C2B domains of synaptotagmin I and II (see FIG. 12).
[0030]
Recently, the C2A domain of synaptotagmin I has been²⁺There were reports that it was sufficient for dependent phospholipid binding. The C2B domain of synaptotagmin is considered to be similar, but the details are unknown. In order to clarify the relationship between inositol phosphate and phospholipid binding to the C2B domain of synaptotagmin, a phospholipid binding test was performed with the GST fusion protein (see FIG. 14). As a result, GST-syt I-C2A and GST-syt II-C2A are Ca²⁺Dependently bound to liposomes (1: 1 (W / W) phosphatidylserine: phosphatidylethanolamine), GST-syt I-C2B and GST-syt II-C2B are Ca²⁺It was found to bind independently. In addition, GST-labfilin 3A (rph) -C2A is Ca²⁺Dependent on phospholipid binding activity, GST-rph-C2B is Ca²⁺It was found that it does not bind even in the presence. That is, the C2A domain of synaptotagmin and labfilin 3A is similar in function to the C2 domain of PKCα,²⁺Although dependent, it is suggested that the C2B domains are different.
When the 327th amino acid of GST-syt II-C2B was changed from lysine to glutamine, phospholipid binding activity was lost, but IP4 binding activity was maintained at 50%.
[0031]
【The invention's effect】
According to the present invention, a peptide that binds to inositol polyphosphate is provided. Since the peptide of the present invention specifically binds to inositol polyphosphate, it is useful as a reagent for studying the action / function of inositol polyphosphate or a purification reagent for inositol polyphosphate. Moreover, according to the screening method of the present invention using the above peptide, it is possible to screen for a substance having physiological activity similar to inositol polyphosphate or a substance that antagonizes inositol polyphosphate. Furthermore, according to the inositol polyphosphate inhibitor of the present invention containing the peptide as an active ingredient, it is possible to inhibit the action of inositol polyphosphate, so that calcium inhibitors, neurotransmitter release inhibitors, hormone release inhibitors It is useful as such.
As described above, the peptide of the present invention is a reagent or purification reagent for studying the action of inositol polyphosphate, a screening reagent for inositol polyphosphate-like substance, a diagnostic reagent for various diseases involving inositol polyphosphate, and treatment (prevention). ) Can be widely used as a drug.
[0032]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to an Example.
Example 1
(1) Purification of IP4 binding protein (IP4BP)
ddY mouse-derived cerebellum 150 g 9 volumes of buffer [0.32 M sucrose, 1 mM EDTA, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), 10 μM leupeptin, 10 μM pepstatin, 1 mM 2-ME, 5 mM Tris-HCl, pH 7.4] And homogenized with a potter homogenizer. The homogenate was centrifuged at 1000 × g for 5 minutes at 2 ° C., and the pellet was washed under the same conditions. The supernatant and the washing solution were combined and centrifuged at 2 ° C. and 100,000 g for 60 minutes to precipitate the P3 + P4 membrane fraction.
[0033]
The membrane fraction was suspended in a buffer (1 mM EDTA, 0.1 mM PMSF, 10 μM leupeptin, 10 μM pepstatin A, 1 mM 2-ME, 50 mM Tris-HCl, pH 8.0), and 20% (w / w) Triton X-100. Was added to adjust the protein concentration to 3.0 mg / ml and the Triton X-100 concentration to 1%. Further, the 100,000 g centrifugal supernatant was applied to a heparin-agarose column (2 × 13.7 cm). The column was 0.2% Triton X-100, 10% glycerol, 10 μM pepstatin A, 10 μM leupeptin, 0.1 mM PMSF, 1 mM 2-ME, 50 mM Tris-HCl, pH 8.0 (referred to as buffer 1). Previously equilibrated with a buffer containing 0.25 M NaCl. The column was then washed with 0.25 M NaCl / Buffer 1 and 0.5 M NaCl / Buffer 1 and IP4BP was eluted with 30 ml of 1 M NaCl / Buffer 1.
[0034]
Further, the eluted fraction was concentrated and applied to a Sepharose column CL-6B (1.0 × 60 cm, equilibrated with 0.2 M NaCl / 1 mM / buffer 1), and the IP4BP fraction was recovered (see FIG. 1a). . The collected fractions are indicated by bars in the figure (the same applies to FIGS. B and c).
The collected fractions were dialyzed against 1 mM EDTA / buffer 1 and then applied to a DE52 column (1.0 × 7.6 cm). The IP4BP fraction was eluted with a 0-0.3M NaCl gradient using buffer 1 containing 1 mM EDTA (see FIG. 1b).
Further, the eluted fraction was purified by rechromatography with Sepharose CL-6B again (see FIG. 1c), and the purified product was stored at −80 ° C. In addition, Table 1 shows the recovery rate in the purification step.
[0035]
[Table 1]

[0036]
Example 2
Analysis by SDS-PAGE
The IP4BP fractions in the above purification step were each subjected to SDS-PAGE (Laemmli method with 3.75-12.5% gradient gel). CBB was used for gel staining. The result is shown in FIG. 2A. In the figure, lane b: 80 μg of solubilized fraction by surfactant; lane c: 30 μg of heparin agarose fraction; lane d: 10 μg of Sepharose CL-6B fraction; lane e: 2 μg of DE-52 fraction; lane f: sepharose The SDS-PAGE pattern of 2 μg of CL-6B rechromatographic fraction is shown. Lane a is a molecular weight marker (× 10^Three200: myosin; 116: β-galactosidase; 92: phosphorylase; 66: bovine serum albumin; 45: ovalbumin.
FIG. 2B shows an SDS-PAGE pattern of 63 to 81 fractions of Sepharose CL-6B rechromatography.
The molecular weight measurement values of IP4BP by SDS-PAGE were 140K and 65K (see FIG. 2C). It was also found that 140K molecules are dimers of 65K molecules.
[0037]
Example 3
[ ^Three H] Measurement of IP4 binding
IP4 binding was measured by PEG precipitation (Maeda, N. ENBO. J.,9, 61-67, 1990). Chromatographic fraction measurement is [^ThreeH] IP4 was added to 50 μl of each fraction to 9.6 nM. The sample was incubated at 0 ° C. for 10 minutes, mixed with 2 μl bovine-gamma globulin (50 mg / ml), 50 μl 30% PEG6000 / 50 mM Tris-HCl (pH 8.0) was added to the sample, and 5 minutes at 0 ° C. Incubated. Next, the 10,000 g centrifugal supernatant was removed, and the precipitate was dissolved in 500 μl of SOLVABLE. Radioactivity was measured by adding 5 ml Aquasol II as a liquid scintillation reagent. Nonspecific binding was measured in the presence of 10 μM unlabeled IP4.
In addition, another IP4 binding assay was used for biochemical characterization. 10 μl [^ThreeH] IP4 (final concentration 4.8 nM), 10 μl gamma globulin (final concentration 50 μg), 10 μl 0.5 M Good buffer, 10 μl inositol phosphate solution (diluted with 50 mM Hepes-NaOH, pH 7.4), sample and The total volume was adjusted to 100 μl with distilled water. After 10 minutes of incubation at 0 ° C., 100 μl of 30% PEG solution was added and measured as described above.
[0038]
Example 4
Identification of IP4BP peptide
Purified IP4BP (about 0.3 mg) obtained in Example 1 was subjected to SDS-PAGE (3.75-12.5% gradient gel), and the separated proteins were transferred to a PVDF membrane to obtain 140K and 65K protein equivalent bands. Was excised from the blot, treated with 0.5 mM NaOH, reduced and alkylated in a microfuge-tube. After washing with 2% acetonitrile, the membrane pieces were treated with lysyl endopeptidase for 24 hours at 37 ° C. The concentration was adjusted such that IP4BP was 1/50 molar in 300 μl of 100 mM Tris-HCl (pH 8.5) containing 7.5% acetonitrile and 0.05% SDS. The membrane piece was removed by centrifugation at 10,000 g for 5 minutes, and the supernatant was lyophilized, dissolved in 0.1% TFA, and HPLC column [Waters 600E, C8 (μBONDASPHERE 300A Waters), column shape 0.39 × 15 cm]. Applied. The gradient of 0-30% 2-propanol / acetonitrile (7: 3) (containing 0.1% TFA) was adjusted to 45 ml or more (0.5 ml / min). The peptide peak was monitored and collected at 215 nm (see FIG. 3A). As shown in the figure, 140K was confirmed to be a 65K dimer.
Next, the main peak of 140K (seven peaks of 1-7 shown in FIG. 3A) was concentrated to 100 μl or less and subjected to a gas-phase sequencer (Applied Biosystem) to determine the amino acid sequence of each. The amino acid sequence of each peptide fragment was compared with the rat-derived synaptotagmin II sequence (see FIG. 3B). As a result, the amino acid sequence matched the rat-derived synaptotagmin II sequence.
[0039]
Example 5
Production of antibodies against C2A domain and C-terminal region peptides of synaptotagmin II
(1) A rat-derived synaptotagmin II C2A domain (amino acid SEQ ID NO: 139-267) and glutathione S-transferase (referred to as GST) fusion protein was expressed in E. coli and purified (by the method of Smith et al .; Gene67, 31-40, 1988). That is, cDNA encoding the C2A domain of rat-derived synaptotagmin II was amplified and prepared by PCR according to a conventional method, subcloned into the pGEX-2T vector, and the fusion protein was expressed in E. coli JM109. The cells were sonicated and treated with Triton X-100 to a final concentration of 1%. Further, the 10,000 g centrifugal supernatant was applied to an affinity column with glutathione-Sepharose CL-6B to purify the fusion protein.
The purified product was analyzed and confirmed by 10% SDS-PAGE, dialyzed into a phosphate buffer (including physiological saline), and injected subcutaneously on the back of a New Zealand white rabbit. The protein solution 1 mg / ml and Freund's complete adjuvant 1 ml were mixed and immunized. Moreover, after boosting between 2 types, the antibody was obtained according to the conventional method.
[0040]
(2) Cysteine was added to the amino terminus of the rat terminal synaptotagmin II C-terminal region peptide (amino acid SEQ ID NO: 409-422) and bound to bovine serum albumin. The albumin is bound to a heterobifunctional crosslinker having a maleimide group that can react with a free sulfhydryl group (sulfo-SMCC-cBSA). 5 mg of C-terminal peptide and 5 mg of sulfo-SMCC-cBSA were mixed in 1 ml of 0.1 M phosphate buffer pH 7.2 (containing 0.1 M EDTA and 0.15 M NaCl) and stirred at room temperature for 3 hours. The reaction was carried out. Unreacted material was removed by gel filtration on a Sephadex G-25 column (under 0.083 M phosphate buffer pH 7.2 and 0.15 M NaCl). The void fraction containing the peptide-cBSA complex was immunized to New Zealand white rabbits in the same manner as described above using aluminum hydroxide as an adjuvant, and antibodies were obtained according to a conventional method.
[0041]
Example 6
Analysis by immunoblotting and immunoprecipitation
(1) Immunoblot method
The solubilized fraction (crude purified protein) and Sepharose CL-6B rechromatographic fraction (purified protein) in Example 1 were subjected to SDS-PAGE (3.75-12.5% Laemmli system), and the protein was applied to a PVDF membrane. After the transfer, it was reacted with the above antibody. The result is shown in FIG. In this figure, lane a is 50 μg of solubilized supernatant; lane b is 3 μg of Sepharose CL-6B rechromatographic fraction (A) stained with amino black (A), reacted with an antibody against the C2A region (B), A reaction with an antibody against the C-terminal region (C) and a reaction with a control antibody (D) are shown. The peroxidase activity used for detection was stained with DAB using the Vectastain ABC kit.
As shown in the figure, it was found that the antibodies against rat C-terminal amino acid sequence (409-422) and C2A region (139-267) of synaptotagmin II derived from rat react with both 140K and 65K.
[0042]
(2) Immunoprecipitation method
Purification of 10 μl of anti-C2A antibody (antibody concentration is IgG, 2, 5, 10 μg / 10 μl / 50 mM Hepes-NaOH, pH 7.4, 0.1 M NaCl, and the antibody was purified with Amersham Protein A column) IP4BP (0.35 μg) was added and incubated in Hepes-NaOH (pH 7.4) buffer containing 0.1 M NaCl for 30 minutes at room temperature. Next, 30 μl of protein A-Sepharose CL-6B suspension (protein A powder 0.3 mg) suspended in the same buffer was added, and the tube was gently shaken at 4 ° C. for 30 minutes. Furthermore, protein A-Sepharose CL-6B particles were precipitated by centrifuging 10,000 g, and 25 μl of the remaining supernatant [^ThreeH] IP4 binding activity was measured by the PEG precipitation method described above. The control experiment was similarly performed using the IgG fraction before rabbit immunization. The result is shown in FIG. In the figure, ○ indicates an anti-C2A antibody, and ● indicates a control antibody. Further,% inhibition (（) indicates the residual activity obtained by subtracting the activity with the anti-C2A antibody from the activity with the control IgG.
As shown in the figure, anti-C2A antibody decreased the IP4 binding activity of purified IP4BP in a dose-dependent manner.
The above results (1) and (2) together with the analysis result of the partial amino acid sequence shown in Example 4, clearly convince that mouse-derived purified IP4BP is a mouse-derived synaptotagmin.
[0043]
Example 7
Characterization of IP4 binding activity of purified IP4BP
FIG. 6 shows the pH dependency of purified IP4BP in IP4 binding. The binding activity is 4.8 nM [^ThreeH] IP4 and 0.35 μg of purified protein were added to 50 mM Mes-NaOH (pH 4.5-6.7; ○), 50 mM Hepes-NaOH (pH 6.2-7.6; □), 50 mM Epps-NaOH (pH 7. 3-8.3; (triangle | delta)) It examined by the buffer solution. Nonspecific binding was determined in the presence of 4.8 μM of label IP4. Samples were incubated at 0 ° C. for 10 minutes and binding was measured by PEG precipitation. As a result, the optimum pH was found to be 5.0.
[0044]
Next, for purified IP4BP [^ThreeH] The saturation of IP4 binding was examined (see FIG. 7). For binding measurement, pH 6.2 (Δ) and pH 7.4 (◯) with 0.35 μg purified protein, or pH 5.0 (□) with 4.8 nM [0.1 μg purified protein].^ThreeH] IP4, various concentrations of cold IP4, and 50 μg gamma globulin were used under 100 μl of 50 mM Mes-NaOH (pH 5.0), 50 mM Hepes-NaOH (pH 6.2 and 7.4). Samples were incubated at 0 ° C. for 10 minutes and binding was measured by PEG precipitation.
As a result, the binding half-maximal reduction was 30 nM at pH 6.2, 40 nM at pH 7.4, and 170 nM at pH 5.0. Scatchard analysis indicates a single bond, with Kd values of 30 nM at pH 6.2 and 7.4, 160 nM at pH 5.0, and Bmax of 2.7 pmol at pH 6.2 and pH 7.4. It was found to be 35 pmol / μg protein at 2.4 pmol and pH 5.0.
[0045]
Next, the specificity of the IP4 binding site was examined by adding five types of inositol polyphosphates. In the presence of various concentrations of IP3 (▲), IP4 (●), Ins 3,4,5,6-P4 (◯), IP5 (■) and IP6 (□), 8nM [^ThreeH] Incubated with IP4. Binding measurements were performed by the PEG precipitation method. The result is shown in FIG. As shown in the figure, IP5 is better than IP4 [^ThreeH] IP4 was strongly replaced. Ins 3,4,5,6-P4 and IP6 inhibited the binding of IP4 by half at 100 nM. However, in the case of IP6, the slope of the replacement curve is relatively sharp and at 10 nM [^ThreeH] Increased binding of IP4. In addition, at 300 nM, [^ThreeH] IP4 binding was strongly suppressed. This means that [^ThreeH] refers to the allosteric-like effect of IP6 on IP4 binding. Other inositol derivatives had low affinity.
[0046]
Example 8
Gene cloning of mouse IP4BP
IP4BP partial amino acid sequence K-3 peptide (VPYQELG; ie, Val-Pro-Tyr-Gln-Glu-Leu-Gly) and K-2 peptide (IHLMQ; ie, Ile-His-) determined in Example 4 5'GTICCITA (TC) CA (AG) GA (AG) (TC) TIGG3 'and 5' (TC) TGCATIA (AG) (AG) TG (AGT) AT3 'corresponding to Leu-Met-Gln) Synthesized as a primer. Total RNA was obtained from Chomczynski and Sacchi (Anal. Biochem.,162, 156-159, 1987), prepared from mouse cerebellum, and poly (A)⁺RNA was selected by oligotex-dT30 (Nippon Roche).
[0047]
PCR amplification was performed for 35 cycles with AMV reverse transcriptase, denatured at 94 ° C. for 2 minutes, annealed at 48 ° C., and subjected to chain extension at 72 ° C. for 3 minutes. The PCR product was extracted with agarose gel electrophoresis and Geneclean II kit (Biol 01), and 2 × 10 2 from a mouse cerebellum-derived cDNA library.⁶Used to screen plaques. Hybridization consists of 50% formamide, 2 × SCC, 0.1% SDS, 100 μg / ml salmon sperm DNA,³²P-label probe (5 × 10^Fivecpm / ml) at 42 ° C. for 12 hours. The hybridizing filter was washed twice at 42 ° C. with 0.1 × SCC and 0.1% SDS. Five positive clones were selected and their cDNAs were cloned into pBluescript KS (-) (Stratagene). Furthermore, the clone was sequenced with BcaBEST Dideoxy Sequencing Kit (Takara Shuzo). The total DNA sequence including a single open reading frame was determined from 5 overlapping clones. The base sequence is shown in SEQ ID NO: 1. The amino acid sequence predicted from the sequence is shown in SEQ ID NO: 2. This sequence was consistent with the partial amino acid sequence described above. In SEQ ID NO: 1, the part with a solid line indicates a partial amino acid sequence that has already been determined.
[0048]
Example 9
Confirmation of IP4BP expression and function
In order to confirm the IP4 binding activity of IP4BP, an expression plasmid (pEF-IP4BP) is prepared, transfected into COS7, and the transformant is cultured to obtain the expression IP4BP / syt II (pEF-IP4BP / syt II). Prepared.
That is, pEF-BOS (Nucleic Acids Res.,18, 5322, 1990) were digested with XbaI, blunted with Klenow fragment and ligated with NotI linkers. All IP4BP / syt II cDNA (1-1590 base pairs) to which NotI linkers were bound was inserted into the NotI site of pEF-BOS. The resulting plasmid (pEF-IP4BP) has the full length IP4BP / syt II cDNA between the human EF1-α promoter and the human G-CSF polyadenylation sequence.
Using the DEAE-dextran method, plasmid pEF-IP4BP and pEF-BOS as a control were transfected into COS7. After transfection, the cells were cultured for 48 hours.
[0049]
After culturing, the cells were homogenized, the homogenate was subjected to SDS-PAGE, and immunoblotted with an anti-IP4BP antibody. The result is shown in FIG. 9A. In the figure, lane 1 is a control (heparin-binding fraction of pEF-BOS-transfected COS7), lane 2 is pEF-IP4BP / syt II, and lane 3 is a mouse cerebellar microsomal fraction.
As shown in the figure, an immunoreactive band was detected in cells transfected with pEF-IP4BP, but not in control cells. The detected band was as low as 60K. Since this molecular weight is the same as that of cerebellum-derived IP4BP / syt II treated with N-glycosidase F, it was proved that the difference in molecular weight was due to the difference in sugar chain.
In addition, control (heparin-binding fraction of pEF-BOS-transfected COS7), pEF-IP4BP / syt II partially purified with heparin-agarose, and cerebellar-derived IP4BP / syt II (each 5.5 μg) [^ThreeH] IP4 binding was compared. The result is shown in FIG. 9B. As shown in the figure, the IP4 binding activity of pEF-IP4BP / syt II was comparable to that derived from the cerebellum.
[0050]
Example 10
Identification of IP4 binding region of IP4BP
In order to identify the IP4 binding region of IP4BP, nine types of fusion proteins of IP4BP and GST excluding the transmembrane region were prepared.
That is, cDNAs encoding various domains of IP4BP / syt II (see FIG. 10A) were grown by PCR. The propagated fragment was subcloned into pGEX-2T and the DNA sequence was confirmed. Preparation of fusion proteins of various domains of IP4BP / syt II and GST (glutathione S-transferase) is described in the literature (Gene67, 31-40, 1988) and expressed in E. coli JM109. The resulting fusion protein was then purified by glutathione-Sepharose 4B (Pharmacia) chromatography and analyzed by SDS-PAGE.
[0051]
In nine types of fusion proteins, the difference in the domain bound to GST is mapped as shown in FIG. 10A. Nine types of fusion proteins are represented by GST-a to GST-i. For example, GST-a represents a fusion protein of GST and domain a.
The IP4 binding properties of these fusion proteins were tested by the method described above. The result is shown in FIG. 10B.
As shown in the figure, it was found that IP4 binding activity exists in the central part of the C2B region. The amino acid sequence corresponded to IHLMQNGKRRLKKKKTTVKKKKTLNPYFNESSFSF of amino acid numbers 315 to 346, that is, the following formula (2).
Ile-His-Leu-Met-Gln-Asn-Gly-Lys-Arg-Leu-Lys-Lys-Lys-Lys-Thr-Thr-Val- Lys-Lys-Lys-Thr-Leu-Asn-Pro-Tyr- Phe-Asn-Glu-Ser-Phe-Ser-Phe- (2)
This region is rich in lysine and is presumed to be important for binding of negatively charged IP4.
In addition, other lysine-rich regions such as C2A region or endogenous polylysine stimulating signal of casein kinase II did not show IP4 binding activity. In addition, this IP4 binding region is a Pep20 peptide (IKKKKTTVKKCTLNPNPYYNESF; ie, Ile-Lys-Lys-Lys-Lys-Thr-Thr-Val-Lys-Lys-Cys-Thr-Leu-Asn-Pro-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr -Asn-Glu-Ser-Phe). Moreover, when this peptide was injected into the squid giant presynaptic terminal, it was shown that the release of neurotransmitters was reversibly suppressed.
[0052]
Example 11
IP4 binding properties of GST-IP4BP
A Scatchard analysis of IP4 binding to purified GST-f (GST-STII-C2B) was performed. The result is shown in FIG. 11A. From this result, Kd was 165 nM and Bmax was 3.8 pmol / μg protein. This Kd value was 4 times higher than that of cerebellum-derived IP4BP (Kd = 40 nM). The GST-STII-C2B has a low affinity, which is presumably because it forms a fusion protein with GST.
[0053]
The binding brain of various inositol polyphosphates to the GST-STII-C2B was measured. The result is shown in FIG. 11B. As shown in the figure, the binding ability is Ins-1,2,3,4,5,6-P6 (IP6)> Ins-1,3,4,5,6-P5 (IP5)> Ins-1. 3, 4, 5-P4 (IP4)> Ins-2, 4,5-P3> Ins-1, 4,5-P3 (IP3)> Ins-1,4-P2. In other words, Ins-1,2,3,4,5,6-P6 and Ins-1,3,4,5,6-P5 are more effective competitors than Ins-1,3,4,5-P4. there were. This indicates that the C2B region of IP4BP / sty II binds not only to IP4 but also to IP5 and IP6.
Similar results were obtained for purified cerebellum-derived IP4BP / sty II except for IP6. Heparin also showed inhibition of binding of IP4 to IP4BP / sty II, but the same was true for GST-STII-C2B.
[0054]
Example 12
Examination of IP4 binding sites
The C2 domain is also known to exist in synaptotagmin I, PKCα and labfilin 3A (Rose John et al. Gene74, 465-471, 1988). Therefore, it was confirmed whether the IP4 binding to the C2B domain is unique to IP4BP / sty II. That is, according to the method described in Example 10, synaptotagmin I, labfilin 3A (rph) and a fusion protein of PKCα protein and GST were prepared, and IP4 binding ability was measured. The result is shown in FIG. In the figure, for example, GST-STI-C2A means a fusion protein of GST and synaptotagmin C2A domain.
As shown in the figure, IP4 binding was found to be specific for the C2B domain of synaptotagmin I, II.
Therefore, amino acid sequences corresponding to the above formula (2) of various organism-derived synaptotagmin C2B domains were compared (see FIG. 13). And it was guessed that the binding site of IP4 exists in GKR (L / I) KKKKKT (T / S) (V / I) KK, that is, in the above formula (1).
That is, in the sequence of FIG. 13, the C-terminal sequence is well conserved and is also found in the synaptotagmin C2A domain, but the synaptotagmin C2A domain does not bind to IP4; the C2 domains of PKCα and labfilin 3A are also IP4 However, since the amino acid sequence of the above formula (1) does not exist in these domains, the peptide represented by the formula (1) was determined to be the IP4 binding site.
[0055]
Example 13
Phospholipid binding properties of C2 domain
If there is a C2A domain of synaptotagmin I,²⁺There are reports that it is sufficient for the binding of dependent phospholipids, but the details are unknown. To elucidate the relationship between inositol phosphate and phospholipid binding to the C2B domain of synaptotagmin, a phospholipid binding test was performed with the GST fusion protein.
That is, liposomes (phosphatidylserine / phosphatidylethanolamine (1: 1, w / w)) and GST fusion protein were incubated for 15 minutes at room temperature in 50 mM Hepes-NaOH (pH 7.4). After centrifugation at 1200 g for 10 minutes, the pellet (phospholipid-binding fraction) and the supernatant (non-binding fraction) were fractionated. Next, both fractions were subjected to 10% SDS-PAGE. The result is shown in FIG. In the figure, lane 1: supernatant-Ca²⁺, Lane 2: Pellet-Ca²⁺, Lane 3: supernatant + 1 mM Ca²⁺, Lane 4: pellet + Ca²⁺A) GST-STII-C2A, b) GST-STII-C2B, c) GST-STII-C2B (327th Lys to Gln), d) GST-STI-C2A, e) GST-STI-C2B, f) GST-C2A + C2B, g) GST-rph-C2A, h) GST-rph-C2B, i) GST.
[0056]
That is, GST-STI-C2A and GST-STII-C2A are Ca²⁺Dependently bound to liposomes (1: 1 (w / w) phosphatidylserine: phosphatidylethanolamine), GST-STI-C2B and GST-STII-C2B are Ca²⁺It has been found to bind to independence. GST-labophilin3A (rph) -C2A is Ca²⁺It has phospholipid binding activity in a dependent manner, but GST-rph-C2B is Ca²⁺It was found that it does not bind even in the presence. That is, the C2A domain of synaptotagmin and labfilin3A is similar in function to the C2 domain of PKCα,²⁺Although dependent, it is suggested that the C2B domains are different. When the 327th amino acid of GST-STII-C2B was changed from lysine to glutamine, phospholipid binding activity was lost, but IP4 binding activity was maintained at 50%. That is, it was found that although IP4 and phosphatidylserine bind to similar regions of the C2B domain, the recognition sites are slightly different.
[0057]
[Sequence Listing]

[0058]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing purification steps of IP4BP by each chromatography.
FIG. 2 is a diagram (A, B) showing the results of SDS-PAGE analysis of each fraction in the IP4BP purification step. In addition, C is a figure which shows the measurement of the molecular weight by SDS-PAGE analysis.
FIG. 3A is a diagram showing HPLC analysis of lysyl endopeptidase degradation products of IP4BP (140k and 65k) separated by SDS-PAGE, and the amino acid sequence of separated fraction (1-7) and rat synaptotagmin II. It is a figure (B) which shows a comparison of an amino acid sequence. In FIG. A, (a) shows a 140k decomposition product, and (b) shows a 65k decomposition product.
FIG. 4 shows the results of immunoblot analysis of crude and purified IP4BP. In the figure, a represents crude IP4BP and b represents purified IP4BP. A is stained with amino black, B is reacted with an antibody against the synaptotagmin C2A region, C is reacted with an antibody against the synaptotagmin C-terminal region, and D is reacted with a control antibody.
FIG. 5 shows the IP4 binding characteristics of IP4BP in the presence of antibodies. In the figure, ◯ indicates the presence of an antibody against the synaptotagmin C2A region, and ● indicates the presence of a control antibody.
FIG. 6 shows the pH dependence of IP4BP on IP4 binding.
[Fig. 7] [IP4BP^ThreeH] IP4 binding saturation and Scatchard analysis.
FIG. 8 is a diagram showing binding characteristics between IP4BP and various inositol polyphosphates. In the figure, ▲ indicates IP3, ● indicates IP4, ◯ indicates Ins 3, 4, 5, 6-P4, ■ indicates IP5, and □ indicates IP6.
FIG. 9 shows the result of immunoblot analysis of IP4BP / syt II (pEF-IP4BP / syt II) expressed by the gene recombination technique (A), and [^ThreeH] IP4 binding ability (B).
In FIG. A, lane 1 is a control, lane 2 is pEF-IP4BP / syt II, and lane 3 is a mouse cerebellar microsomal fraction.
FIG. 10 is a diagram showing the position of the synaptotagmin domain bound to GST in the fusion protein of GST and various synaptotagmin domains prepared in Example 10, and a diagram showing the IP4 binding ability of the fusion protein (FIG. 10). B).
FIG. 11 is a diagram (A) showing the results of Scatchard analysis of the fusion protein GST-STII-C2B, and a diagram (B) showing the binding ability to various inositol polyphosphates.
FIG. 12 is a diagram showing IP4 binding ability of fusion proteins of GST and various C2 domains.
FIG. 13 is a diagram comparing amino acid sequences of regions corresponding to formula (2) of synaptotagmin C2B domains derived from various organisms. In the figure, MII is mouse synaptotagmin II, MI is mouse synaptotagmin I, HI is human synaptotagmin I, BI is bovine synaptotagmin I, RA is shibiraei synaptotagmin A, RB is shibirae synaptotagmin B, SI is ikasynaptotagmin I, DI is Drosophila synaptotagmin I and CI indicate nematode synaptotagmin I.
FIG. 14 shows phospholipid binding properties for GST-various C2 domain fusion proteins.

Claims (4)

A peptide having the following amino acid sequence, peptides having binding inositol tetrakis phosphate, against inositol pentakis phosphate or inositol hexakis phosphate.
Gly-Lys-Arg-X ₁ -Lys-Lys-Lys-Lys-Thr-X ₂ -X ₃ -Lys-Lys
(Wherein X ₁ represents Leu or Ile, X ₂ represents Thr or Ser, and X ₃ represents Ile or Val) Peptide, a peptide according to claim 1, wherein the peptide constituting the C2B domain of synaptotagmin I or II. A method for screening an inositol polyphosphate-like substance or an inositol polyphosphate antagonist, comprising using the peptide according to claim 1 or 2. An inositol polyphosphate inhibitor containing the peptide according to claim 1 or 2 as an active ingredient.

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