JPH02223593A - Human carbastatin-like polypeptide - Google Patents
Human carbastatin-like polypeptideInfo
- Publication number
- JPH02223593A JPH02223593A JP1107388A JP10738889A JPH02223593A JP H02223593 A JPH02223593 A JP H02223593A JP 1107388 A JP1107388 A JP 1107388A JP 10738889 A JP10738889 A JP 10738889A JP H02223593 A JPH02223593 A JP H02223593A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- polypeptide
- sequence
- formula
- calpain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 83
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 70
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 70
- 150000001413 amino acids Chemical class 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000011347 resin Substances 0.000 abstract description 14
- 229920005989 resin Polymers 0.000 abstract description 14
- -1 9- fluorenylmethoxycarbonyl group Chemical group 0.000 abstract description 4
- 125000006239 protecting group Chemical group 0.000 abstract description 3
- 208000002177 Cataract Diseases 0.000 abstract description 2
- 208000032839 leukemia Diseases 0.000 abstract description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 3
- 210000004899 c-terminal region Anatomy 0.000 abstract 2
- 239000000463 material Substances 0.000 abstract 1
- 201000006938 muscular dystrophy Diseases 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000010532 solid phase synthesis reaction Methods 0.000 abstract 1
- 235000001014 amino acid Nutrition 0.000 description 31
- 108010032088 Calpain Proteins 0.000 description 26
- 102000007590 Calpain Human genes 0.000 description 26
- 230000000694 effects Effects 0.000 description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 102100035037 Calpastatin Human genes 0.000 description 10
- 108010044208 calpastatin Proteins 0.000 description 10
- ZXJCOYBPXOBJMU-HSQGJUDPSA-N calpastatin peptide Ac 184-210 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCSC)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(O)=O)NC(C)=O)[C@@H](C)O)C1=CC=C(O)C=C1 ZXJCOYBPXOBJMU-HSQGJUDPSA-N 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 101001019513 Homo sapiens Calpastatin Proteins 0.000 description 4
- 150000008064 anhydrides Chemical class 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 102000045597 human CAST Human genes 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- 102000010831 Cytoskeletal Proteins Human genes 0.000 description 2
- 108010037414 Cytoskeletal Proteins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 1
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000014824 Crystallins Human genes 0.000 description 1
- 108010064003 Crystallins Proteins 0.000 description 1
- 102000005927 Cysteine Proteases Human genes 0.000 description 1
- 108010005843 Cysteine Proteases Proteins 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 101150111020 GLUL gene Proteins 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- LCPYQJIKPJDLLB-UWVGGRQHSA-N Leu-Leu Chemical group CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC(C)C LCPYQJIKPJDLLB-UWVGGRQHSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000014750 Phosphorylase Kinase Human genes 0.000 description 1
- 108010064071 Phosphorylase Kinase Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108010085012 Steroid Receptors Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical group OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 108010091798 leucylleucine Proteins 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000003365 myofibril Anatomy 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 102000005969 steroid hormone receptors Human genes 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、生体内で種々の生理活性を有するヒトカルパ
インの活性を阻害する新規なポリペプチドに関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel polypeptide that inhibits the activity of human calpain, which has various physiological activities in vivo.
カルパスタチンは、カルシウム依存性のシスティンプロ
テアーゼとして知られているカルパインを特異的に阻害
する細胞内蛋白質である。Calpastatin is an intracellular protein that specifically inhibits calpain, which is known as a calcium-dependent cysteine protease.
カルパスタチンは高等動物の各種の組織に広く分布し、
細胞が刺激を受けることによって動員され、活性化され
たカルパインの作用を調節していると考えられている。Calpastatin is widely distributed in various tissues of higher animals,
It is thought that it is mobilized when cells receive stimulation and regulates the action of activated calpain.
カルパインの生体における役割としては、プロティンキ
ナーゼＣ１ホスホリラーゼキナーゼＢなどのキナーゼ系
統酵素の限定分解による活性化、細胞骨格蛋白質の分解
、増殖因子やホルモンのレセプターの分解などが知られ
ている。Calpain's roles in living organisms are known to include activation through limited degradation of kinase enzymes such as protein kinase C1 phosphorylase kinase B, degradation of cytoskeletal proteins, and degradation of growth factor and hormone receptors.
カルパスタチンは、これらのカルパインの作用を特異的
に阻害することにより、カルパインの過剰反応を調節し
ている。したがってカルパイン−カルパスタチン系のバ
ランスの維持は、病態と密接に関連している。Calpastatin regulates the excessive reaction of calpain by specifically inhibiting these calpain actions. Therefore, maintenance of the balance of the calpain-calpastatin system is closely related to pathological conditions.
例えば、筋ジストロフイー疾患においては、カルパイン
のレベルが高まっており、筋原繊維やニューロフィラメ
ントの分解がカルパインによって促進されることが、発
症と密接に関わっていると推定されている。また、成人
型白血病ウィルスに感染した細胞では、カルパイン活性
及びインターロイキン２のレセプター活性が異常に高ま
っていることが知られている。これは、カルパインが細
胞骨格蛋白質に作用してレセプター活性を変化させ、そ
の結果、増殖因子などに対する細胞の異常な反応を生み
出し、病因となることを推定させる〔生化学、第５７巻
、第１２０２頁（１９８５））。更にカルパインは眼水
晶体蛋白質を切断することが知られており、その過剰反
応が白内障に関係していることが示唆される。For example, in muscular dystrophic diseases, the level of calpain increases, and it is assumed that the promotion of decomposition of myofibrils and neurofilaments by calpain is closely related to the onset of the disease. Furthermore, it is known that calpain activity and interleukin 2 receptor activity are abnormally increased in cells infected with adult leukemia virus. This suggests that calpain acts on cytoskeletal proteins to change receptor activity, resulting in abnormal cellular responses to growth factors and the like, leading to pathogenesis [Biochemistry, Vol. 57, No. 1202] (1985)). Furthermore, calpain is known to cleave ocular lens proteins, and its overreaction has been suggested to be related to cataracts.
一方、情報伝達に必要なステロイド−レセプター複合体
の安定化物質が、カルパスタチンであるという報告〔ジ
ャーナル オブ バイオロジカル ケミストリー（Ｊｏ
ｕｒｎａｌ ｏｆ ＢｉｏｌｏｇｉｃａｌＣｈｅｍｉ
ｓｔｒｙ ） 、第２６０巻、第２６０１頁（１９８５
）：］や、高血圧ラットにおけるカルパスタチンレベル
の異常な減少も報告されている〔バイオケミカルアンド
バイオフィジカル リザーチ コミュニケーションズ
（Ｂｉｏｃｈｅｍｉｃａｌ ａｎｄ Ｂｉｏｐｙｓｉｃ
ａｌＲｅｓｅａｒｃｈ Ｃｏｍｍｕｎｉｃａｔｉｏｎｓ
） 、第１４５巻、第１２８７頁（１９８７）：］。On the other hand, it has been reported that calpastatin is a substance that stabilizes the steroid-receptor complex necessary for information transmission [Journal of Biological Chemistry (Jo
urnal of Biological Chemistry
stry), Volume 260, Page 2601 (1985
):] and an abnormal decrease in calpastatin levels in hypertensive rats [Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)].
alResearch Communications
), Volume 145, Page 1287 (1987): ].
このようにカルパスタチンは、カルパインの作用を調節
する内在性の蛋白質インヒビターとして、カルパインの
過剰作用が原因と考えられる種々の疾患に対して有効な
治療剤となることが期待される。As described above, calpastatin, as an endogenous protein inhibitor that regulates the action of calpain, is expected to be an effective therapeutic agent for various diseases thought to be caused by excessive action of calpain.
カルパスタチンは高等動物の各種組織に広く分布し、ブ
タ赤血球や心筋、ウサギ骨格筋及びヒト赤血球などから
の精製が報告されている〔ザ バイオケミカル ジャー
ナル（Ｔｈｅ Ｂｉ。Calpastatin is widely distributed in various tissues of higher animals, and purification from pig red blood cells, cardiac muscle, rabbit skeletal muscle, and human red blood cells has been reported [The Biochemical Journal (The Bi.
ｃｈｅｍｉｃａｌ Ｊｏｕｒｎａｌ） 、第２３５巻、
第９７頁（１９８６）、ジャーナル オブ バイオケミ
ストリ＝ （Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｃｈｅｍｉ
ｓｔｒｙ ） 、第９５巻、第１６６１頁（１９８４）
、ジャーナル オブ バイオケミストリ、第９２巻、第
２０２１頁（１９８２））。更に、ブタ及びウサギのカ
ルパスタチンのｃＤＮＡクローニングにより、アミノ酸
の一次構造が解明された〔バイオケミストリー （Ｂｉ
ｏｃｈｅｍｉｓｔｒｙ ） ）第２７巻１第９６４頁（
１９８Ｂ）、プロシーディングズ オブザ ナショナル
アカデミ−オブ サイエンシーズ オブ ザ Ｕ Ｓ
Ａ （Ｐｒｏｃｅｅｄｉｎｇｓｏｆ ｔｈｅ Ｎａ
ｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ ５ｃｉｅｎｃｅ
ｓ、 Ｕ Ｓ Ａ ）、第８４巻、第３５９０頁（１９
８７）］。その結果、カルパスタチンの基本構造は共通
であり、約１４０アミノ酸残基からなる４つの繰り返し
機能単位（ドメイン１−４）とＮ末側の非相同性の配列
（ドメインＬ）よりなることがわかった〔フエブスレタ
ーズ（ＦＩＥＢＳ Ｌｅｔｔｅｒｓ ）、第２２３巻、
第１７４頁（１９８７））。Chemical Journal), Volume 235,
No. 97 (1986), Journal of Biochemistry
stry), Volume 95, Page 1661 (1984)
, Journal of Biochemistry, Volume 92, Page 2021 (1982)). Furthermore, cDNA cloning of pig and rabbit calpastatin elucidated the primary structure of amino acids [Biochemistry (Bi
ochemistry)) Volume 27, No. 1, Page 964 (
198B), Proceedings of the National Academy of Sciences of the U.S.
A (Proceeding of the Na
tional Academy of 5sciences
s, USA), Volume 84, Page 3590 (19
87)]. As a result, it was found that calpastatin has a common basic structure, consisting of four repeating functional units (domains 1-4) consisting of approximately 140 amino acid residues and a non-homologous sequence on the N-terminal side (domain L). [FIEBS Letters, Volume 223,
No. 174 (1987)).
ヒトカルパスタチンの一次構造に関しては、まず本発明
者らがドメイン１−４の配列を明らかにし〔特願昭６３
−１０９１１１号〕、次いで全配列を解明した〔特願昭
６３−２０７２８３号〕。本発明の目的は、ヒトカルパ
スタチンの活性を有し、種々の応用に至便な鎖長の短い
ポリペプチドを提供することにある。Regarding the primary structure of human calpastatin, the present inventors first clarified the sequence of domains 1-4 [Patent application No. 63
-109111], and then the entire sequence was elucidated [Japanese Patent Application No. 63-207283]. An object of the present invention is to provide a short-chain polypeptide that has the activity of human calpastatin and is convenient for various applications.
本発明を概説すれば、本発明はヒトカルパスタチン様ポ
リペプチドに関する発明であって、下記式■で示される
ことを特徴とする。To summarize the present invention, the present invention relates to a human calpastatin-like polypeptide, and is characterized by being represented by the following formula (2).
Ｘ−Ｔｈｒ−Ｔｙｒ−１１ｅ−Ｇｌｕ−Ｇｌｕ−Ｌｅｕ
−Ｇｌｙ−Ｌｙｓ八ｒへ−Ｇ］ ｕ−Ｖａ］−Ｔｈｒ−
Ｉ Ｉｅ−Ｐｒｏ−Ｐｒｏ−ＬｙｓＴｙｒ−八ｒｇ−Ｙ
・・・
（Ｉ）〔式中Ｘは下記式：
Ａｓｐ−Ｐｒｏ−Ｍｅｔ−３ｅｒ−３ｅｒで表される配
列又はそのＮ末端からアミノ酸若しくはペプチドが欠失
した配列を示し、Ｙは下記式：
％式％
で表される配列又はそのＣ末端からアミノ酸若しくはペ
プチドが欠失した配列を示す〕本明者らはペプチド合成
法によりヒトカルパスタチンドメイン内の１８アミノ酸
よりなるポリペプチド〔前記式■に示されるＸ及びＹが
欠失した１８アミノ酸ポリペプチド二以下１８アミノ酸
ポリペプチドと略す〕、２１アミノ酸よりなるポリペプ
チド〔前記式■で表されるポリペプチドで、式中、Ｘが
欠失し、Ｙ ＝ Ｇｌｕ−Ｌｅｕ−Ｌｅｕであるような
ポリペプチド：以下２１アミノ酸ポリペプチドと略す］
及び２６アミノ酸よりなるポリペプチド〔前記式■で表
されるポリペプチドで、式中、Ｘ−八ｓｐ−Ｐｒｏ−Ｍ
ｅｔ−８ｅｒ−３ｅｒかつＹ ＝ Ｇｌｕｌ、ｅ ｕ
−Ｌ ｅ ｕであるようなポリペプチド：以下２６アミ
ノ酸ポリペプチドと略す〕を合成しカルパイン阻害活性
を有することを見出し、この知見に基づいて本発明を完
成させた。以下本発明を具体的に説明する。X-Thr-Tyr-11e-Glu-Glu-Leu
-Gly-Lys8r-G]u-Va]-Thr-
I Ie-Pro-Pro-LysTyr-8rg-Y
...
(I) [wherein X represents the sequence represented by the following formula: Asp-Pro-Met-3er-3er or a sequence in which an amino acid or peptide is deleted from its N-terminus, and Y represents the following formula: % formula % The present inventors synthesized a polypeptide consisting of 18 amino acids within the human calpastatin domain using a peptide synthesis method. 18-amino acid polypeptide with Y deleted (abbreviated as 18-amino acid polypeptide below 2)], polypeptide consisting of 21 amino acids [polypeptide represented by the above formula (2), in which X is deleted, and Y = Glu- Polypeptide that is Leu-Leu: hereinafter abbreviated as 21 amino acid polypeptide]
and a polypeptide consisting of 26 amino acids [a polypeptide represented by the above formula (1), in which X-8sp-Pro-M
et-8er-3er and Y = Glul, e u
-L eu (hereinafter abbreviated as 26 amino acid polypeptide)] was found to have calpain inhibitory activity, and based on this knowledge, the present invention was completed. The present invention will be specifically explained below.
ヒトカルパスタチンはそのｃＤＮ八クリクローニングり
アミノ酸の一次構造が明らかにされ、約＼
１４０アミノ酸残基よりなる４つの繰り返し機能単位（
ドメイン１−４）とＮ末側に非相同性の配列（ドメイン
Ｌ）を有することが見出されている〔特願昭６：３−２
０７２８３号〕。ドメイン１−４は各ドメイン単独でカ
ルパスタチンの活性を有しているが、このうちドメイン
１が最も強い活性を有しているので、このドメインにつ
いて更に詳細な解析を行った。その結果、ドメイン１は
前記１８アミノ酸ポリペプチドまで短縮しても活性を示
すことを見出した。The primary structure of human calpastatin was revealed by cloning its cDN and revealed that it consists of four repeating functional units consisting of approximately 140 amino acid residues (
It has been found that it has a non-homologous sequence (domain L) on the N-terminal side with domain 1-4) [Patent Application No. 6:3-2
No. 07283]. Domains 1 to 4 each have calpastatin activity on their own, but among these domains, domain 1 has the strongest activity, so this domain was analyzed in more detail. As a result, it was found that domain 1 showed activity even when shortened to the 18 amino acid polypeptide.
そこでこの１８アミノ酸ポリペプチド、２１アミノ酸ポ
リペプチド、及び２６アミノ酸ポリペプチドを実際に化
学合成し、そのカルパイン阻害活性を調べた。ポリペプ
チドの化学合成はＦｍｏｃ（９−フルオレニルメトキシ
カルボニルポリアミド）合成法に従って行った〔ザ ジ
ャーナルオブ オーガニック ケミストリー（Ｔｈｅ
Ｊｏｕｒｎａｌ ｏｆ Ｏｒｇａｎｉｃ Ｃｈｅｍｉｓ
ｔｒｙ）、第３７巻、第３４０４頁（１９７２）、ジャ
ーナル オブ ザ ケミカルソサエティー、ケミカル
コミュニケーションズ（Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ
Ｃｈｅｍｉｃａｌ ５ｏｃｉｅｔｙ、 Ｃｈｅｍｉｃ
ａｌ Ｃｏｍｍｕｎｉｃａｔｉｏｎｓ ） 、第１３
巻、第５３７頁（１９７Ｂ）、インターナショナル ジ
ャーナル オブ ペプチド アンド プロティン リサ
ーチ（Ｉｎｔｅｒｎａｔｉｏｎａｌ 、Ｉｏｕｒｎａｌ
ｏｆ Ｐｅｐｔｉｄｅ ａｎｄ Ｐｒ。Therefore, these 18 amino acid polypeptides, 21 amino acid polypeptides, and 26 amino acid polypeptides were actually chemically synthesized, and their calpain inhibitory activity was investigated. Chemical synthesis of the polypeptide was performed according to the Fmoc (9-fluorenylmethoxycarbonyl polyamide) synthesis method [The Journal of Organic Chemistry (The
Journal of Organic Chemistry
try), Volume 37, Page 3404 (1972), Journal of the Chemical Society, Chemical
Communications (Journal of the
Chemical 5ociety, Chemical
al Communications), No. 13
Vol. 537 (197B), International Journal of Peptide and Protein Research.
of Peptide and Pr.
ｔｅｉｎ Ｒｅ５ｅａｒｃｈ ） 、第１３巻、第３５
頁（１９７９））。tein Research), Volume 13, No. 35
(1979)).
合成されたポリペプチドは確かにカルパインの阻害活性
を有しており、１８アミノ酸ポリペプチドだけでカルパ
スタチン様活性を示すことが明らかになった。また、２
１アミノ酸ポリペプチド及び２６アミノ酸ポリペプチド
でもカルパスタチン様活性を示すことが明らかになった
。以上述べてきたごとく、本発明により、ヒトカルパス
タチン様活性を示す新規な１８アミノ酸ポリペプチド、
２１アミノ酸ポリペプチド及び２６アミノ酸ポリペプチ
ドを提供することが可能となった。It has been revealed that the synthesized polypeptide does indeed have calpain inhibitory activity, and that only the 18 amino acid polypeptide exhibits calpastatin-like activity. Also, 2
It was revealed that a 1-amino acid polypeptide and a 26-amino acid polypeptide also exhibit calpastatin-like activity. As described above, according to the present invention, a novel 18 amino acid polypeptide exhibiting human calpastatin-like activity,
It has become possible to provide a 21 amino acid polypeptide and a 26 amino acid polypeptide.
次に本発明の実施例を示すが、これは本発明を限定する
ものではない。Next, examples of the present invention will be shown, but these are not intended to limit the present invention.
実施例１１８アミノ酸ポリペプチド、２１アミノ酸ポリ
ペプチド及び２６アミノ酸ポリ
ペプチドの合成
（原料） Ｆｍｏｃ−Ａｓｐ（ＯＢｕｔ）−ＰＰＰＰ
ｍｏｃ−Ｐｒｏ−ＰＰＰ
Ｆｍｏｃ−Ｍｅｔ−ＰＰＰ
Ｆｍｏｃ−３ｅｒ （日ｕｔ）−ＤＩＩＢＴＰｍｏｃ−
Ｔｈｒ（Ｂｕｔ）−ＤＨＢＴＦｍｏｃ−Ｔｙｒ （Ｂｕ
ｔ） −ＰＰＰＦｍｏｃ−４１ｅ−ＰＰＰ
Ｐｍｏ叶旧ｕ （Ｂｕｔ） −ＰＰＰ
Ｆｍｏｃ−Ｌｅｕ−ＰＰＰ
Ｆｍｏｃ−Ｇ］ｙ−ＰＰＰ
Ｆｍｏｃ−Ｌｙｓ （Ｂｏｃ）−ＰＰＰＦｍｏｃ−Ａｒ
ｇ （Ｍｔｒ） −ＤｔｌＢＴＦｍｏｃ−Ｖａｌ−ＰＰ
Ｐ
Ｆｍｏｃ−Ａｒｇ（Ｍｔｒ）−Ｏｆｆ
Ｆ ｍ ｏ ｃ −Ｌ ｅ ｕ −Ｄ Ｈなおりｕｔは
ｔ−ブチルを、ＰＦＰはペンタフルオロフェニルを、Ｄ
ＨＢＴは１−オキソ−２−ヒドロキシジヒドロベンゾト
リアジンを、Ｍｔｒは４−メトキシ−２，３，６−）リ
メチルフェニルスルホニルを、Ｂｏｃはｔ−ブチルオキ
シカルボニルを示す。Example 1 Synthesis of 18-amino acid polypeptide, 21-amino acid polypeptide and 26-amino acid polypeptide (raw materials) Fmoc-Asp(OBut)-PPPP
moc-Pro-PPP Fmoc-Met-PPP Fmoc-3er (Japan UT)-DIIBTPmoc-
Thr(But)-DHBTFmoc-Tyr(Bu
t) -PPPFmoc-41e-PPP Pmo Kano (But) -PPP Fmoc-Leu-PPP Fmoc-G]y-PPP Fmoc-Lys (Boc)-PPPFmoc-Ar
g (Mtr) -DtlBTFmoc-Val-PP
P Fmoc-Arg(Mtr)-Off Fmoc-Leu-D H naoriut is t-butyl, PFP is pentafluorophenyl, D
HBT represents 1-oxo-2-hydroxydihydrobenzotriazine, Mtr represents 4-methoxy-2,3,6-)limethylphenylsulfonyl, and Boc represents t-butyloxycarbonyl.
（樹脂） ペプシンＫＡ［ミリゲン（Ｍ ｉ ］ Ｉ
１Ｇｅｎ）社製〕
１．１８アミノ酸ポリペプチドのＣ末端のアミノ酸であ
るＦ ｍ ｏ ｃ −Ｌ ｅ ｕ −Ｄ Ｈの樹脂及び
２１アミノ酸ポリペプチドと２６アミノ酸ポリペプチド
のＣ末端のアミノ酸であるＰｍｏｃ−Ｌｅｕ−叶の樹脂
への結合及びペプチド鎖の延長
（１） ＣＦｍｏｃ−Ａｒｇ （Ｍｔｒ） ） ２０
無水物及び（ＦｍｏｃＬｅｕ） ２０無水物の合成
７３０ｍｇのＰｍｏｃ−Ａｒｇ （Ｍｔｒ）−叶を塩化
メチレン５ｍｌ！に溶解し、ジシクロへキシルカルボジ
イミド １１０ｍｇを添加し、水浴中４０分間かくはん
後、ジシクロヘキシル尿素をろ過により除いてろ液を減
圧下で、蒸発乾固した。４２０ｍｇのＦｍｏｃ−Ｌｅｕ
−ＯＨについても同様に処理した。(Resin) Pepsin KA [Milligen (M i ) I
1Gen)] 1. Fmoc-Leu-DH resin, which is the C-terminal amino acid of a 18-amino acid polypeptide, and Pmoc-Leu-DH, which is the C-terminal amino acid of a 21-amino acid polypeptide and a 26-amino acid polypeptide. Leu-binding to leaf resin and extension of peptide chain (1) CFmoc-Arg (Mtr) ) 20
Synthesis of anhydride and (FmocLeu) 20 anhydride 730 mg of Pmoc-Arg (Mtr)-Leu in 5 ml of methylene chloride! After adding 110 mg of dicyclohexylcarbodiimide and stirring in a water bath for 40 minutes, dicyclohexylurea was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure. 420mg Fmoc-Leu
-OH was treated in the same manner.
（２） 〔Ｆｍｏｃ−Ａｒｇ （Ｍｔｒ） ：］ ２
０及び（Ｆｍｏｃ−Ｌｅｕ） ２０と樹脂（ペプシンＫ
Ａ）の結合
ＣＦｍｏｃ−Ａｒｇ （Ｍｔｒ） ） ２０無水物 ７
２０ｍｇを樹脂が湿る程度のジメチルホルムアミド
（ＤＭＰ）に溶かし、４−ジメチルアミノピリジン１１
ｍｇの存在下、樹脂１ｇと約２時間反応させた。反応終
了後ＤＭＦ１０ｄ、次いで塩化メチレン１０誦で洗浄し
乾燥させて、アミノ酸分析にて導入率を確認した。(2) [Fmoc-Arg (Mtr):] 2
0 and (Fmoc-Leu) 20 and resin (pepsin K
A) Bond of CFmoc-Arg (Mtr) ) 20 anhydride 7
Dissolve 20mg of 4-dimethylaminopyridine 11 in dimethylformamide (DMP) to wet the resin.
1 g of resin for about 2 hours. After the reaction was completed, the mixture was washed with 10 ml of DMF and then 10 ml of methylene chloride, dried, and the introduction rate was confirmed by amino acid analysis.
（Ｆｍｏｃ−Ｌｅｕ） ２０無水物４１０ｍｇについて
も同様に処理した。(Fmoc-Leu) 20 anhydride (410 mg) was treated in the same manner.
（３） ペプチド鎖の延長
次に生成したＰｍｏ叶Ａｒｇ （Ｍｔｒ）−ペプシンＫ
Ａ樹脂をペプチドシンセサイザー用カラム（ミリゲン社
製）に充てんし、ペプチドシンセサイザー（ミリゲン社
製）を用いて１８アミノ酸ポリペプチドのＣ末端側から
順番にＦｍｏｃ−Ｔｙｒ（Ｂｕｔ）−ＰＰＰ 、 Ｆｍ
ｏｃ−Ｌｙｓ（Ｂｏｃ）ＰＰＰ、以下１８番目のＦｍｏ
ｃ−Ｔｈｒ （Ｂｕｔ） −ＤＨＢＴまでを加えて結合
させ、１８アミノ酸ポリペプチドを合成した。また、Ｆ
ｍｏｃ−Ｌｅｕ−ペプシンＫＡ樹脂については、２１ア
ミノ酸ポリペプチド及び２６アミノ酸ポリペプチドのＣ
末端側から順番にＦｍｏｃ−Ｌｅｕ−ＰＢＰ、 Ｆｍｏ
ｃ−Ｇｌｕ（Ｂｕｔ）−ＰＰＰ 、以下２１番目のＦｍ
ｏｃ−Ｔｈｒ （Ｂｕｔ）叶ＢＴまでを加えて結合させ
、２１アミノ酸ポリペプチドを、２６番目のＦｍｏｃ−
八ｓｐ （ＯＢｕｔ）ＰＰＰまでを加えて結合させ、２
６アミノ酸ポリペプチドを合成した。合成終了後、ペプ
チドの結合した樹脂を塩化メチレン１０ｍ１（４回）、
ｔ−アミルアルコール１０社（２回）、酢酸１０ｍ１！
（２回）、ジエチルエーテル１０ｍ２（４回）で洗浄し
た。(3) PmoArg (Mtr)-pepsin K generated after peptide chain extension
A peptide synthesizer column (manufactured by Milligen) was filled with resin A, and using a peptide synthesizer (manufactured by Milligen), Fmoc-Tyr(But)-PPP and Fm were sequentially synthesized from the C-terminus of the 18 amino acid polypeptide.
oc-Lys(Boc)PPP, 18th Fmo below
c-Thr (But) -DHBT was added and bound to synthesize an 18 amino acid polypeptide. Also, F
For moc-Leu-pepsin KA resin, C of 21 amino acid polypeptide and 26 amino acid polypeptide
Fmoc-Leu-PBP, Fmo in order from the terminal side
c-Glu(But)-PPP, 21st Fm below
oc-Thr (But) up to Kano BT are added and bonded, and the 21 amino acid polypeptide is combined with the 26th Fmoc-
Add up to 8 sp (OBut) PPP and combine, 2
A 6 amino acid polypeptide was synthesized. After the synthesis, the peptide-bound resin was treated with 10 ml of methylene chloride (4 times).
10 t-amyl alcohols (2 times), 10ml of acetic acid!
(2 times) and 10 m2 of diethyl ether (4 times).
２、 樹脂並びに保護基の除去
合成完了済みの樹脂に２０％ピペリジンＤＭＦを加え１
時間放置後、Ｄ Ｍ Ｆ １０ｍ１２、次いで塩化メチ
レン１０ｍ１２で洗浄し乾燥した。それにトリフルオロ
酢酸（ＴＦＡ）：エタンジチオール：チオアニソール−
９０：５：５の混合液１０ｍ１を加え、３時間放置した
。次にＴＦＡ濃度が５％になるように水を加えてから、
ヘキサン１０艷、次いでエーテル１０−で洗浄した。2. Removal of resin and protective groups Add 20% piperidine DMF to the synthesized resin 1
After standing for a period of time, it was washed with 10 ml of DMF, then 10 ml of methylene chloride, and dried. And trifluoroacetic acid (TFA): ethanedithiol: thioanisole-
10 ml of a 90:5:5 mixture was added and left to stand for 3 hours. Next, add water so that the TFA concentration is 5%, and then
Washed 10 times with hexane and then 10 times with ether.
洗浄後、水をエバポレーターで除去しＩＮ酢酸１ｍｌに
溶かし、一部を高速液体クロマトグラフィー（カラム：
ＹＭＣ−０ＤＳ５Ｃ，、、移動相：０．０５％Ｔ Ｆ
Ａ／Ｈ２０−０，０５％ＴＦＡ／ア七ト二トリル）を
行い、溶出された各ピークフラクションすべてについて
アミノ酸分析で調べた。アミノ酸組成が合うフラクショ
ンを分取し、保護基の遊離の確り忍のため高速原子衝撃
マススペクトルで分子量を調べた。その結果、１８アミ
ノ酸ポリペプチドが３２ｍｇ、 ２１アミノ酸ポリペプ
チドが４０ｍｇ、 ２６アミノ酸ポリペプチドが５０ｍ
ｇ得られた。After washing, water was removed using an evaporator, dissolved in 1 ml of IN acetic acid, and a portion was subjected to high performance liquid chromatography (column:
YMC-0DS5C, Mobile phase: 0.05% TF
A/H20-0.05% TFA/A7Tonitrile) was performed, and all of the eluted peak fractions were examined by amino acid analysis. Fractions with matching amino acid compositions were collected, and their molecular weights were examined using high-speed atom impact mass spectroscopy to confirm the release of the protecting groups. As a result, the 18 amino acid polypeptide was 32 mg, the 21 amino acid polypeptide was 40 mg, and the 26 amino acid polypeptide was 50 mg.
g was obtained.
実施例２ 合成したポリペプチドのカルパイン阻害活性
の検出
カルパインの阻害活性は、以下の方法に従って行ったし
ジャーナル オブ バイオケミストリー、第９５巻、第
１７５９頁（１９８４）］。Example 2 Detection of calpain inhibitory activity of synthesized polypeptide The inhibitory activity of calpain was determined according to the following method (Journal of Biochemistry, Vol. 95, p. 1759 (1984)).
サンプル１０μβ、１ ｍｇ／−カルパイン溶液２．５
μＡ、２％カゼイン／イミダゾール溶液４０μｌ、６ｍ
ｇ／ｍシスティン溶液２０μｌと水とを混合し、全量で
１８０μｐとした。次に３０℃で５分間予熱した後、５
０ｍＭ塩化カルシウム溶液を２０μβ加えて、３０℃で
１５分間インキュベートした。その後５％トリクロロ酢
酸を２００μβ加えて反応を停止し、１２００Ｏｒｐｍ
で１０分間遠心し、上清を回収した。その上清の２８０
ｎｍの吸光度を測定して、これよりカルパイン阻害活性
を算出した。Sample 10 μβ, 1 mg/- calpain solution 2.5
μA, 40 μl of 2% casein/imidazole solution, 6 m
20 μl of g/m cysteine solution and water were mixed to give a total volume of 180 μp. Next, after preheating at 30℃ for 5 minutes,
20μβ of 0mM calcium chloride solution was added and incubated at 30°C for 15 minutes. After that, 200 μβ of 5% trichloroacetic acid was added to stop the reaction, and the temperature was adjusted to 1200 Orpm.
The mixture was centrifuged for 10 minutes and the supernatant was collected. 280 of the supernatant
The absorbance at nm was measured, and the calpain inhibitory activity was calculated from this.
その結果、表１に示したように実施例１で合成したポリ
ペプチドは確かに、カルパインの阻害活性を有しており
、１８アミノ酸ポリペプチド、２１アミノ酸ポリペプチ
ド及び２６アミノ酸ポリペプチドはカルパスタチン様活
性を示すことが明らかになった。As a result, as shown in Table 1, the polypeptide synthesized in Example 1 did indeed have calpain inhibitory activity, and the 18-amino acid polypeptide, the 21-amino acid polypeptide, and the 26-amino acid polypeptide were calpastatin-like. It was revealed that it showed activity.
参考例１１８アミノ酸ポリペプヂドのＮ末端よりアミノ
酸若しくはペプチドが
欠失したポリペプチドのカルパイ
ン阻害活性の検出
（１）Ｎ末端欠失ポリペプチドの合成
前記実施例１に示す手段でＰｍｏｃ−Ａｒｇ （Ｍｔｒ
）ペプシンＫＡ樹脂に、１８アミノ酸ポリペプチドのＣ
末端側から順番にＦｍｏｃ−Ｔｙｒ （Ｂｕｔ）ＰＰＰ
、 Ｆｍｏｃ−Ｌｙｓ（Ｂｏｃ）−ＰＰＰ以下、１１番
目のＰｍｏｃ−Ｌｙｓ （Ｂｏｃ）−ＰＰＰまでを結合
させ、合成したポリペプチド、１２番目のＰｍｏｃ−Ｇ
ｌｙ−ＰＰＰまでを結合させ、合成したポリペプチド、
１３番目のＰｍｏｃ−Ｌｅｕ−ＰＰＰまでを結合させ、
合成したポリペプチド、１４番目のＰｍｏｃ−Ｇｌｕ（
Ｂｕｔ）−ＰＰＰまでを結合させ、合成したポリペプチ
ド、１５番目のＦｍｏｃ−Ｇｌｕ（Ｂｕｔ）−ＰＰＰま
でを結合させ、合成したポリペプチド、１６番目のＰｍ
ｏｃ−１１ｅ−ＰＰＰまでを結合させ、合成したポリペ
プチド、及び１７７番目ＦｍｏｃＴｙｒ （Ｂｕｔ）−
ＰＰＰまでを結合させ、合成したポリペプチドを得た。Reference Example 1 Detection of calpain inhibitory activity of a polypeptide in which an amino acid or peptide is deleted from the N-terminus of a 118-amino acid polypeptide (1) Synthesis of N-terminally deleted polypeptide Pmoc-Arg (Mtr
) C of an 18 amino acid polypeptide on Pepsin KA resin.
Fmoc-Tyr (But)PPP in order from the terminal side
, a polypeptide synthesized by binding Fmoc-Lys (Boc)-PPP and up to the 11th Pmoc-Lys (Boc)-PPP, a 12th Pmoc-G
A polypeptide synthesized by binding up to ly-PPP,
Combine up to the 13th Pmoc-Leu-PPP,
The synthesized polypeptide, the 14th Pmoc-Glu (
Fmoc-Glu(But)-PPP up to the synthesized polypeptide, the 15th Fmoc-Glu(But)-PPP was bonded up to the synthesized polypeptide, the 16th Pm
The polypeptide synthesized by binding up to oc-11e-PPP, and the 177th FmocTyr (But)-
A synthesized polypeptide was obtained by binding up to PPP.
（２） カルパイン阻害活性の検出
これらの合成ポリペプチドを用いて、前記実施例２に示
す手段でカルパイン阻害活性を検出した。その結果、こ
れらＮ末端欠失ポリペプチドのカルパイン阻害活性は皆
無若しくは極めて微弱でその活性の強さが測定できない
程であることが明らかになった。(2) Detection of calpain inhibitory activity Using these synthetic polypeptides, calpain inhibitory activity was detected by the method shown in Example 2 above. As a result, it was revealed that these N-terminally deleted polypeptides had no or very weak calpain inhibitory activity, to the extent that the strength of the activity could not be measured.
以上の結果から、本発明により新規なヒトカルパスタチ
ン様活性を示す１８アミノ酸ポリペプチド及びそのＮ末
端、Ｃ末端を延長したポリペプチドが提供された。この
新規１８アミノ酸ポリペプチド及びそのＮ末端、Ｃ末端
を延長したポリペプチドは、カルパインの過剰作用が原
因と考えられる種々の疾患に対しての効果がある。From the above results, the present invention provides a novel 18 amino acid polypeptide exhibiting human calpastatin-like activity and a polypeptide with its N-terminus and C-terminus extended. This novel 18 amino acid polypeptide and its N- and C-terminally extended polypeptides are effective against various diseases thought to be caused by excessive action of calpain.
Claims (1)
スタチン様ポリペプチド。 【遺伝子配列があります】・・・（ I ） 〔式中Ｘは下記式： 【遺伝子配列があります】 で表される配列又はそのＮ末端からアミノ酸若しくはペ
プチドが欠失した配列を示し、Ｙは下記式： 【遺伝子配列があります】 で表される配列又はそのＣ末端からアミノ酸若しくはペ
プチドが欠失した配列を示す〕[Claims] 1. A human calpastatin-like polypeptide represented by the following formula I. [There is a gene sequence]...(I) [In the formula, X represents the following formula: [There is a gene sequence] or a sequence in which an amino acid or peptide is deleted from its N-terminus, and Y is the following: Formula: [There is a gene sequence] or a sequence in which an amino acid or peptide is deleted from the C-terminus]
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1990624400 DE69024400T2 (en) | 1989-04-28 | 1990-04-20 | Human calpastatin-like polypeptide |
| EP19900304237 EP0395309B1 (en) | 1989-04-28 | 1990-04-20 | Human calpastatin-like polypeptide |
| US07/852,343 US5189144A (en) | 1989-04-28 | 1992-03-17 | Human calpastatin-like polypeptide |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63-280333 | 1988-11-08 | ||
| JP28033388 | 1988-11-08 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02223593A true JPH02223593A (en) | 1990-09-05 |
| JP2796835B2 JP2796835B2 (en) | 1998-09-10 |
Family
ID=17623541
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1107388A Expired - Fee Related JP2796835B2 (en) | 1988-11-08 | 1989-04-28 | Human calpastatin-like polypeptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2796835B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010033912A3 (en) * | 2008-09-19 | 2011-06-16 | Henry Ford Health System | Methods, systems, and compositions for calpain inhibition |
| CN118909046A (en) * | 2024-09-14 | 2024-11-08 | 陈锦济生物医学技术(广州)有限公司 | Plant polypeptide and application thereof in preparation of cataract inhibiting medicine |
-
1989
- 1989-04-28 JP JP1107388A patent/JP2796835B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| PROC NATL ACAD SCI=1987US * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010033912A3 (en) * | 2008-09-19 | 2011-06-16 | Henry Ford Health System | Methods, systems, and compositions for calpain inhibition |
| CN118909046A (en) * | 2024-09-14 | 2024-11-08 | 陈锦济生物医学技术(广州)有限公司 | Plant polypeptide and application thereof in preparation of cataract inhibiting medicine |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2796835B2 (en) | 1998-09-10 |
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