JP2018511344A - 接着性ペプチド - Google Patents
接着性ペプチド Download PDFInfo
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- JP2018511344A JP2018511344A JP2018501837A JP2018501837A JP2018511344A JP 2018511344 A JP2018511344 A JP 2018511344A JP 2018501837 A JP2018501837 A JP 2018501837A JP 2018501837 A JP2018501837 A JP 2018501837A JP 2018511344 A JP2018511344 A JP 2018511344A
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Abstract
Description
[X1−X2−X3−X4−X5]n
前記X1は任意のアミノ酸で、
X2、X3及びX4は、L、V、I、E及びAのうちいずれか一つのアミノ酸であり、各々同じまたは異なり、X5は、KまたはRのアミノ酸である。
[X6−X7−X8−X9−X10−X11]n
X6は、F、Y及びWのうちいずれか一つのアミノ酸で、
X7は、KまたはRのアミノ酸で、
X8は、A、M及びIのうちいずれか一つのアミノ酸で、
X9は、L、M及びGのうちいずれか一つのアミノ酸で、
X10は、任意のアミノ酸で、
X11は、C、S及びTのうちいずれか一つのアミノ酸である。
前記化学式Iにおいて、前記X2、X3またはX4のうち一つ以上は欠如されてもよい。
X12 1−15
[X1−X2−X3−X4−X5]n
X1は、極性の非荷電された任意のアミノ酸であり、
X2、X3及びX4は、L、V、I、E及びAのうちいずれか一つのアミノ酸であり、各々同じまたは異なり、
X5は、KまたはRのアミノ酸である。
[X6−X7−X8−X9−X10−X11]n
X6は、F、Y及びWのうちいずれか一つのアミノ酸で、
X7は、KまたはRのアミノ酸で、
X8は、A、M及びIのうちいずれか一つのアミノ酸で、
X9は、L、M及びGのうちいずれか一つのアミノ酸で、
X10は、任意のアミノ酸で、
X11は、C、S及びTのうちいずれか一つのアミノ酸である。
前記nは1または2である。
<実施例1.接着性ペプチドの製造>
細胞接着には、ペプチドを予めコートする方法とコートせず細胞培養液に直接添加して培養する二種類の方法で観察し、一致する結果を示した。コート方法を取る場合、PBSまたは、ウシ胎児血清を含まない細胞培養培地に一定濃度のペプチドを添加して、常温、30分間コートした後、溶液を除去した後、細胞を添加して多様な時間にかけて接着を測定した。また、培養培地にペプチドを添加する場合、細胞を懸濁する時、一定水準のペプチドを共に添加してよく懸濁した後培養皿に移した。細胞接着測定は次の通りに行われた。細胞接着は、ペプチドを添加しなかった場合を陰性対照群に、PBSそしてpoly−L−lysinをコートした場合を陽性対照群にして光学顕微鏡を介して通常の観察、アクチンフィラメント構造及び粗い細胞膜境界形態観察のために、F−actin染色及び共焦点顕微鏡分析、そして接着に対する定量化のために細胞の消費量またはDNA量を測定した。消費量は、CCK−8(Dojindo)を利用して、吸光度を測定して、DNA量は、ピコグリーン定量キット(Picogreenassay kit(Life Technologies))を製造者の方法のとおり利用して蛍光値を測定した。接着された細胞だけを定量化するために、接着されなかった細胞は捨ててPBSでさらに2回洗浄して表面に付いている細胞だけを消費量またはDNAを定量に使用した。
まず、タンパク質合成を阻害すると知られているEDTA、シクロヘキサミド(CHX)(10μM、37℃1時間)またはGAGで細胞を処理した後上述したような接着能を実験した。
次は、非タンパク質物質の関与の有無を調べて、その対象に細胞表面の細胞外メトリックス(Extracellualrmatrix,ECM)に多量含まれているプロテオグリカンとの関連性を分析した。
プロテオグリカンを構成する主な成分であるヘパラン硫酸(heparan sulfate)、ヘパリン(heparin)、コンドロイチン硫酸(chondroitin sulfate)、デルマタン硫酸(dermatansulfate)、ケラタン硫酸(keratan sulafate)等のグリコアミノグリカン(glycoaminoglycan)(GAG)は、ECMを構成する主な成分でもあるが、ECM構造の保存性(structuralintegrity of ECM)を維持して細胞の形態を維持すると共に、細胞の接着、細胞の極性(cellpolarity)を調節する。ECMが有するこのような機能は、環境に対する細胞の適応を誘導すると共に複雑な一連の代謝過程と直接的に関連していて、様々な生理学的役割を調節する複合的機能を有する。プロテオグリカンに対する本願ペプチドの分子親和力は、(i)アフィニティークロマトグラフィー、(ii)精製されたGAG成分を利用した競争的結合を介した細胞接着調査、(iii)GAG成分を含むECMを特異的に分解する酵素を処理してペプチドによる細胞接着促進の抑制調査、以上の三つの方法を介して検証した。酵素処理の場合、ヒアルロニダーゼ(hyaluronidase(Sigma))、コラゲナーゼ(collagenase(Sigma))を処理した。精製されたGAGは、hyaluronan(Sigma)、コンドロイチン硫酸(chondroitinsulfate(Sigma))、ヘパリン(heparin(Sigma))、ヘパラン硫酸(heparan sulfate(Sigma))を各々使用した。
(3)RGDと比較
Claims (20)
-
前記X1は、任意のアミノ酸で、
X2、X3及びX4は、L、V、I、E及びAのうちいずれか一つのアミノ酸であり、各々同じかまたは異なり、
X5は、KまたはRのアミノ酸であり、
前記nは、1乃至5の整数であり、二つ以上含む場合、各ペプチドのアミノ酸配列は、同じかまたは異なり、前記アミノ酸は、D−またはL−型の天然または非天然アミノ酸またはその誘導体である、
前記化学式Iのペプチド。 - 前記X1の任意のアミノ酸は、S、T、C、P、NまたはQである、請求項1に記載のぺプチド。
- 前記X2−X3−X4は、LVV、AAA、EEE、LVG、LVA、LVL、LVV、LLA、LLL、またはLLVである、請求項1または2に記載のペプチド。
- 前記化学式Iのペプチドは、
QLVVK(配列番号1)、QEEEK(配列番号2)、QAAAK(配列番号3)、NLVVK(配列番号4)、またはSLVVK(配列番号5)のいずれか一つである、請求項1乃至3のいずれか一項に記載のペプチド。 - 前記化学式Iのペプチドは、
X6は、F、Y及びWのうちいずれか一つのアミノ酸で、
X7は、KまたはRのアミノ酸で、
X8は、A、M及びIのうちいずれか一つのアミノ酸で、
X9は、L、M及びGのうちいずれか一つのアミノ酸で、
X10は、任意のアミノ酸で、
X11は、C、S及びTのうちいずれか一つのアミノ酸で、
前記X7及びX8または前記X10及びX11のうち一つ以上は欠如されてもよく、
前記化学式Iで前記X2、X3またはX4のうち一つ以上は欠如されてもよく、
前記nは、1または2であり、
前記化学式IIのペプチドは、前記化学式Iのペプチドのアミノ末端(N−末端)またはカルボキシ末端(C−末端)、またはN−末端及びC−末端の全てに連結される、
一つ以上の前記化学式IIのペプチドをさらに含む、請求項1乃至4のいずれか一項に記載のペプチド。 - 前記化学式IIのペプチドは、
FRALPC(配列番号6)、FREEPC(配列番号7)、FRVVPC(配列番号8)、FEALPC(配列番号9)、YRALPC(配列番号10)、WRALPC(配列番号11)、FRALP(配列番号12)、FRAL(配列番号13)、またはFRPC(配列番号14)のいずれか一つである、請求項1乃至5のいずれか一項に記載のペプチド。 - 前記ペプチドは、
一つ以上の前記化学式IIIのペプチドを、そのNまたはC末端、或いはN及びC末端にさらに含む、請求項1または5に記載のペプチド。 - 前記正荷電されたアミノ酸はKまたはRで、
前記負荷電されたアミノ酸はDまたはEである、請求項7に記載のペプチド。 - 前記ペプチドは、
RQLVVK(配列番号15);
FRALPC(配列番号6);
FRALPCRQLVVK(配列番号16);
RQLVVKFRALPC(配列番号17);
RQLVVKFRALPCRQLVVKFRALPC(配列番号18);
RQLVVKFRALP(配列番号19);
RQLVVKFRAL(配列番号20);
KQLVVKFRALPC(配列番号21);
RQKFRALPC(配列番号22);
RQEEEKFRALPC(配列番号23);
RQAAAKFRALPC(配列番号24);
RQLVVKFRPC(配列番号25);
RQLVVKFREEPC(配列番号26);
RQLVVKFRVVPC(配列番号27);
RQEEEKFREEPC(配列番号28);
EQLVVEFEALPC(配列番号29);
RQLVVKYRALPC(配列番号30);
RQLVVKWRALPC(配列番号31);
RNLVVKFRALPC(配列番号32);
RSLVVKFRALPC(配列番号33);
R-(QLVV)2-KFRALPC(配列番号34);
R-(QLVV)3-KFRALPC(配列番号35);
R-(QLVV)4-KFRALPC(配列番号36);
RQLVVK-(FRALPC)2(配列番号37);
(R)2-QLVVKFRALPC(配列番号38);
(R)5-QLVVKFRALPC(配列番号39);
(R)10-QLVVKFRALPC(配列番号40);または
(R)15-QLVVKFRALPC(配列番号41)のいずれか一つである、請求項7または8に記載のペプチド。 - 前記ペプチドは、
RQLVVKFRALPC(配列番号17);
KQLVVKFRALPC(配列番号21);
RNLVVKFRALPC(配列番号32);
RSLVVKFRALPC(配列番号33);
RQVVVKFRALPC(配列番号42);
RQIVVKFRALPC(配列番号43);
RQAVVKFRALPC(配列番号44);
RQEVVKFRALPC(配列番号45);
RQLLVKFRALPC(配列番号46);
RQLIVKFRALPC(配列番号47);
RQLAVKFRALPC(配列番号48);
RQLEVKFRALPC(配列番号49);
RQLVLKFRALPC(配列番号50);
RQLVIKFRALPC(配列番号51);
RQLVAKFRALPC(配列番号52);
RQLVEKFRALPC(配列番号53);
RQAAAKFRALPC(配列番号24);
RQEEEKFRALPC(配列番号23);
RQLVVRFRALPC(配列番号54);
RQLVVKYRALPC(配列番号30);
RQLVVKWRALPC(配列番号31);
RQLVVKFKALPC(配列番号55);
RQLVVEFEALPC(配列番号56);
RQLVVKFRLLPC(配列番号57);
RQLVVKFRILPC(配列番号58);
RQLVVKFRVLPC(配列番号59);
RQLVVKFRELPC(配列番号60);
RQLVVKFRAAPC(配列番号61);
RQLVVKFRAIPC(配列番号62);
RQLVVKFRAVPC(配列番号63);
RQLVVKFRAEPC(配列番号64);
RQLVVKFRVVPC(配列番号27);
RQLVVKFREEPC(配列番号26);
RQEEEKFREEPC(配列番号28);
RQEEEEFEEEPC(配列番号65);
RQLVVKFRALXC(配列番号66);
RQLVVKFRALPS(配列番号67);
RQLVVKFRALPT(配列番号68);または
RQLVVKFRALPX(配列番号69)のいずれか一つである、請求項7または8に記載のペプチド。 - 前記ペプチドのC−末端は、非反応性基で置き換えられたものである、請求項1乃至10のいずれか一項に記載のペプチド。
- 前記ペプチドは、薬物、標識物質、または標的物質をさらに含む、請求項1乃至10のいずれか一項に記載のペプチド。
- 請求項1乃至10のいずれか一項に記載のペプチドを含む、バイオコンジュゲート用組成物。
- 請求項1乃至10のいずれか一項に記載のペプチドを含む、生体由来物質または非生体由来物質に対する接着用組成物。
- 請求項1乃至10のいずれか一項に記載のペプチドをコードする、核酸。
- 請求項15に記載の核酸を含む、ベクター。
- 請求項16に記載のベクターで形質転換された、細胞。
- 請求項1乃至10のいずれか一項に記載のペプチドをバイオコンジュゲート用物質として用いる使用方法。
- 請求項1乃至10のいずれか一項に記載のペプチドを生体由来物質または非生体由来物質に対する接着用物質として用いる使用方法。
- 請求項1乃至10のいずれか一項に記載のペプチドを細胞付着のために用いる使用方法。
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