JP3910909B2 - A novel cathepsin B-like cysteine protease derived from pink shrimp - Google Patents

Description

位置は配列番号１の位置番号を示す。3'-及び5'-AUAPプライマーは3'- 及び 5'-RACE kitに含まれているプライマーで同じ配列である。NはA/C/G/Tを表す。
【００２５】
＜1st strand cDNAの作成＞
肝膵臓より抽出した全RNA1μgから、3'RACE System for rapid amplification of cDNA ends (GIBCO BRL)を用いて、1st strand cDNAを作成した。
【００２６】
＜3'RACE＞
上記の1st strand cDNA約1μgを鋳型にして、20 pmolのプライマー（P1及び3'-AUAP）、20 nmol dNTP、10μlの10 x Ex taq buffer、1Uの Ex taq DNA polymerase(Takara)を含む100μlの反応液でPCRを行った。PCRは、DNAサーマルサイクラー(PCR system 9700、PERKIN ELMER)を用いて、94℃で3分間処理後、94℃で30秒間の熱変性 、60℃で30秒間のアニーリング、72℃で1分間の伸張反応のサイクルを30回行い、最後の伸張工程は5分間延長し、終了させた。さらに、nestedプライマーP2を用いて、同様の反応条件でPCRを行った。増幅産物は1％アガロースゲル電気泳動により分離後、エチジウムブロマイド染色により検出した（図.1A）。次に、増幅産物をpGEM-T Easyベクターに挿入して、サブクローニングし、3'末端側の塩基配列を決定した。
【００２７】
＜5'RACE＞
3'RACE法で決定した配列を基に、表１に示したアンチセンス鎖のプライマーP3、P4及びP5を作成し、5'RACE System for rapid amplification of cDNA ends, version 2 (GIBCO BRL)を用いて、プロトコールに従って5'RACE法を行った（図.1A）。得られたPCR断片を、3'RACE法と同様にサブクローニングし、5'末端側の塩基配列を決定した。
【００２８】
＜全長の単離と配列決定＞
全長のcDNAを増幅するために、5'RACEで得られた塩基配列の5'末端側よりプライマーP6を作成した。上述の1st strand cDNAからプライマーP6と3'-AUAPを用いて、ホッコクアカエビ・カテプシンBをコードする全長のcDNAを単離し（図.1B）、塩基配列を決定した。決定した塩基配列と演繹アミノ酸配列を図２に示した。
【００２９】
全長はポリAテールを含めて1,159塩基からなり、開始コドン(ATG)から終始コドン(TGA)までの987bpの翻訳領域を含んでいた。ポリアデニレーションシグナルAATAAAは3'非翻訳領域の1110-1115塩基目に存在した。
【００３０】
＜ホッコクアカエビ・カテプシンBの一次構造＞
ホッコクアカエビ・カテプシンB遺伝子は、328アミノ酸残基をコードしており、推定された15残基のシグナルペプチド、60残基のプロペプチド及び253残基の成熟型酵素領域を含んでいた（図2）。演繹アミノ酸配列をBLAST searchによる相同性検索に供した結果、哺乳類のカテプシンBと高い相同性を示した。
【００３１】
図.3はBLASTPで高い相同性を示した哺乳類のカテプシンB及び無脊椎動物のカテプシンB様システインプロテアーゼとともに、ホッコクアカエビ・カテプシンBをアラインメントした図である。プレプロ領域は一次構造よりもむしろ二次構造において保存されているので、それらは一次構造の相同性が低いため、成熟型酵素領域（図.3B）から分離して（図.3A）アラインメントした。
【００３２】
パパインスーパーファミリーに属するシステインプロテアーゼは配列に基づいて、プロ領域にERFNINモチーフを持つカテプシンL様酵素などのグループと、それを持たないカテプシンB様酵素の2つのサブグループに分けられている(Karrer et al., 1993)。ホッコクアカエビ・カテプシンBには、ERFNINモチーフが存在しなかった。また、ほとんどのシステインプロテアーゼにおいて、保存されているGNFDモチーフ(Vernet et al., 1995)は、ホッコクアカエビ・カテプシンBにも存在した（図.3A）。
【００３３】
ホッコクアカエビ・成熟型カテプシンBはヒトカテプシンBと最も高い相同性（53％）を示した。その他の生物のカテプシンBまたはカテプシンB様酵素とのアミノ酸配列の相同性を表2に示す。
【００３４】
【表２】

【００３５】
ホッコクアカエビ・成熟型カテプシンBは、一般的にシステインプロテアーゼの特徴を示すアミノ酸残基のすべて、及びカテプシンBに特徴的なアミノ酸残基を含んでいた。すなわち、ホッコクアカエビ・成熟型カテプシンBは、触媒基であるCys29、His199及びAsn219（番号はヒトの成熟型カテプシンBの位置番号を示す。以下同様）、オキシアニオンホールを形成するGln23及びGly27、基質の主鎖と相互作用する部位の近傍であるGly73、Gly74及びTrp221、N末端付近に存在するPro2を含んでいた（図.3B）。カテプシンBは、パパインスーパーファミリーに属する他のシステインプロテアーゼに比べ、エキソペプチダーゼ活性を示すという点で特徴的である。この特徴を示すために重要な役割を担うoccludingループ（105塩基目から126塩基目）（Musil et al., 1991）は、ホッコクアカエビ・カテプシンBにも存在していた。さらに、このループに存在し、S2'サブサイト（Schechter and Bergerの表記法）を構成する2つの連続するヒスチジン残基(His110及びHis111)は、保存されていた（図.3B）。また、ヒト・カテプシンBにおいて6つのジスルフィド結合を形成する12のシステイン残基は、ホッコクアカエビ・カテプシンBにおいても保存されていた（図.3B）。
【００３６】
＜ホッコクアカエビ・カテプシンBの系統関係＞
ホッコクアカエビ・カテプシンB、パパイン及びパパインスーパーファミリーに属する典型的なカテプシンについて、プレプロ領域を除いた成熟型酵素の系統樹を作成した。系統樹は近隣結合法により、PHYLIP package, version 3.5c (Felsenstein, 1993) を用いて構築した。系統樹においても、ホッコクアカエビ・カテプシンBは、カテプシンBの系統群に分類された（図.4）。
【００３７】
＜ホッコクアカエビ・カテプシンBの構造特性＞
ホッコクアカエビ・成熟型カテプシンBの分子量の計算値は28,108であり、これまでに知られているカテプシンB及びそのホモログの分子量の範囲と良く一致した。ホッコクアカエビ・成熟型カテプシンBは、カテプシンBの中で最も高い負の電荷を持っていた。等電点の理論値は、他のホモログに比べて顕著に低く、pI 4.19であった。また、Kyte and Doolittle (1982)の方法で計算したhydropathicityは、‐0.487と推定された。電荷、等電点及びhydropathicity値の結果から、ホッコクアカエビ・カテプシンBは、低温適応した水圏生物の典型的な構造特性を示した。
【００３８】
＜ノーザンブロット解析＞
Sambrook (1989)らの方法に従って、以下の操作を行った。上述の各組織の全RNA (10μg)を電気泳動後、Hybond-N+膜に転写した後、65℃で１晩ハイブリダイゼーションを行った。3'末端領域のDNA断片369塩基（696-1064塩基目）をPCRにより増幅し、Random Primer DNA Labeling kit Ver.2.0 (Takara)を用いて、〔α-32P〕dCTPにより標識した。ノーザンブロット解析を行った結果、肝膵臓において圧倒的に強いシグナルが検出された（図５）。
【００３９】
＜発現系の構築＞
大腸菌を用いた系では、pET39b（Novagen）に本発明の遺伝子を挿入し、大腸菌BL21（DE3）株に導入して、発現誘導することにより、発現することができた。SDS-PAGEにより、目的の分子量を持つ融合蛋白質が発現されたことを確認した。
【００４０】
酵母を用いた系では、本遺伝子を挿入した酵母発現ベクターpPICZ α (Invitrogen)をエレクトロポレーション法により、宿主酵母 P. Pastoris KM71H 株に導入し、高濃度（2mg/ml）のzeocinを含む培地で増殖できる形質転換体を選別した。単一コロニーの形質転換株（KM71H株）を125mlのGY（glycerol-complex medium,pH<3.0）に接種し、30℃で72時間振盪培養した。次に、この培養液を遠心により集菌して、15mlのMM（minimal methanol medium,pH<3.0）に再懸濁した。メタノール添加を約24時間ごとに繰り返し、26℃で120時間振盪培養した。SDS-PAGEによる解析で、目的の蛋白質に相当するサイズのバンドが検出された。次に、ゼラチンザイモグラフィーで活性を確認したところ、目的の酵素に相当するサイズのバンドが検出された。また、カテプシンBの特異的基質であるZ-Arg-Arg-MCA（Bachem）を用いて活性を確認できた。
【００４１】
REFERENCES
Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle.
Karrer, K.M., Peiffer, S.L., DiTomas, M.E., 1993. Two distinct gene subfamilies within the family of cysteine protease genes. Proc. Natl. Acad. Sci. USA. 90, 3063-3067.
Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132.
Musil, D., Zucic, D., Turk, D., Engh, R.A., Mayr, I., Huber, R., Popovic, T., Turk, V., Towatari, T., Katunuma, N., et al., 1991. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 10, 2321-2330.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor laboratory press, New York.
Schechter, I., Berger, A., 1967. On the size of the active site in proteinases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157-162.
Vernet, T., Berti, P.J., de Montigny, C., Musil, R., Tessier, D.C., Menard, R., Magny, M.C., Storer, A.C., Thomas, D.Y., 1995. Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing. J. Biol. Chem. 270, 10838-10846.
【００４２】
[実施例２] ホッコクアカエビ肝膵臓粗抽出液のカテプシンB酵素活性。
＜粗抽出液の調製＞
粗抽出液は、‐80℃で凍結保存した肝膵臓から調製した。凍結した組織を部分解凍し、2倍量の150mM NaClを含む50mMリン酸緩衝液(pH 6.5)とともに、ポリトロンホモジナイザーを用いて5分間ホモジナイズした。1/3倍量のテトラクロロメタンをゆっくりと加えて攪拌後、混合液を遠心分離することにより、脱脂を行った。脱脂された上清を粗抽出液として用いた。
【００４３】
＜カテプシンB活性の測定＞
カテプシンBの特異的基質であるZ-Arg-Arg-MCAを用いて粗抽出液の活性を測定した。2 mM EDTA, 2 mM DTT, 0.05% Triton X-100を含む100 mM酢酸緩衝液(pH 5.5)中、基質の終濃度100μMで反応を行った。遊離するAMCの蛍光強度を励起波長370 nm、蛍光波長460 nmで測定した。測定は5回繰り返し行った。標準曲線を作成して遊離するAMCを定量し、25℃で、1分間に1μmolのAMCを遊離させる酵素量を1Uとした。その結果、粗抽出液は、438.1±9.1 mUと高いカテプシンB活性を示した。この活性は、システインプロテアーゼの特異的阻害剤であるE-64により阻害された。また、セリンプロテアーゼの特異的阻害剤であるPMSFはほとんど影響しなかった。以上のことから、ホッコクアカエビ肝膵臓には蛋白質レベルでもカテプシンBが存在することが示唆された。
【００４４】
省略記号： AMC, 7-amino-4-methylcoumarin; DTT, dithiothreitol; E-64, L-trans-epoxysuccinyl-leucyl-agmatine; MCA, 4-methyl-7-coumarylamide; PMSF, phenylmethylsulfonyl fluoride, Z, benzyloxycarbonyl.
【００４５】
【発明の効果】
本発明により、ホッコクアカエビ由来新規カテプシンBが提供される。本酵素はホッコクアカエビ肝膵臓より、または本酵素をコードする遺伝子を導入し、形質転換した宿主細胞を培養することにより得ることができ、食品分野、化粧品分野、医薬分野など幅広い分野において有用に利用することができる。
【００４６】
【配列表】

【図面の簡単な説明】
【図１】 ホッコクアカエビ・カテプシンBをコードするcDNAのクローニングの概要を示した図である。(A)3'-及び5'-RACEに用いたプライマーの位置の模式図とそれぞれのPCR産物の電気泳動図を示した。(B)シングルPCRによる全長cDNAの単離を模式的に示した図とPCR産物の電気泳動図である。M, DNAサイズマーカー； 3', 3'RACE産物； 5', 5'RACE産物； F, 全長cDNA。
【図２】 ホッコクアカエビ・カテプシンBのヌクレオチド配列と演繹アミノ酸配列を示した図である。塩基配列の太字は、開始コドン(ATG)および終始コドン(TGA)を示し、下線は推定されるポリアデニレーションシグナル(AATAAA)およびポリAテールを示す。アミノ酸配列の白い長方形とグレーの長方形で囲った領域は、それぞれシグナルペプチドとプロペプチドを示し、白抜きは、活性部位のシステイン、ヒスチジンおよびアスパラギン残基を示す。
【図３】 ホッコクアカエビ・カテプシンBをその他の生物のカテプシンBまたはカテプシンB様システインプロテアーゼとともにアラインメントした図である。省略記号は、Pb, ホッコクアカエビ; CtB, cathepsin B; Hs, Homo sapiens; Bt, Bos taurus; Sp, Sarcophaga peregrina; Dm, Drosophila melanogaster; Bm, Bombyx mori; Sm, Schistosoma mansoniを示す。ホッコクアカエビ・カテプシンBと同一のアミノ酸残基及び類似のアミノ酸残基をそれぞれ黒とグレーで示した。(A)プレプロ領域のアラインメントを示した図である。(B)成熟型酵素のアラインメントを示した図である。
なお、配列上の数字は、ヒトカテプシンＢにおける位置番号を示している。
また、Ｓ１，Ｓ２，Ｓ３，Ｓ１’、Ｓ２’はバインディングポケットを構成するアミノ酸残基を表す。アスタリスク＊の付いた番号は、触媒基のシステイン、ヒスチジン、アスパラギンを表す。
黒丸印は、ヒトカテプシンＢにおいてジスルフィド結合を形成するシステイン残基を、四角で囲った部分はoccluding loopを示す。
【図４】 図.3に示したすべてのカテプシンB（太字で示す）と様々なシステインプロテアーゼの成熟型酵素の配列に基づき、近隣結合法により無根系統樹を作成した図である。省略記号は、Ct, cathepsin; Rn, Rattus norvegicus; Lma, Leishmania major; Lme, Leishmania mexicana; PAM, percent accepted mutationを示す。各分岐点の数値はブートストラップ検定の値を示す。
【図５】 ホッコクアカエビの各組織におけるカテプシンBをコードするmRNAのノーザンブロット解析の結果を示した図である。全RNA10μgを、0.9%アガロースゲルで電気泳動後、Hybond-N+ membraneにブロッティングした。レーン1、筋肉；レーン2、肝膵臓；レーン3、腸。RNAサイズマーカーを左側に示した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to polynucleotides encoding cathepsin B-like cysteine proteases, novel proteases newly identified from pink shrimp, polypeptides encoded thereby, and the use of such polynucleotides and polypeptides.
[0002]
[Prior art]
Proteases are one of the most important enzymes that hydrolyze peptide bonds in proteins and are widely distributed in microorganisms, plants and animals and are involved in a wide variety of biological processes. In addition, proteases are roughly classified into four families based on catalytic groups: aspartic acid-, cysteine-, serine-, and metallo-proteases. The molecular mechanism of action of these enzymes has been extensively studied.
[0003]
The range of application of protease is wide-ranging, and for example, it is used for food modifiers, detergents, cosmetic raw materials, beer clarifiers, leather tanning agents, and pharmaceuticals.
[0004]
Cathepsin B (EC 3.4.22.1) and its homolog belonging to the papain superfamily of cysteine proteases have so far been identified and sequenced from several organisms. These are listed below.
Aedes aegypti (yellow fever mosquito), Ancylostoma caninum (dog hookworm), Bos taurus (cow), Caenorhabditis elegans, Gallus gallus (chicken), Haemonchus contortus, Homo sapiens (human), Leishmania major, Leishmania mexicana, mouse musculus ), Rattus norvegicus (Norway rat), Sarcophaga peregrina, Triticum aestivum (bread wheat), Schistosoma mansoni
[0005]
The most well-studied is mammalian cathepsin B, but parasite cathepsin B homologs are also widely studied. Parasite cathepsin B homologs have been shown to have several properties that differ from mammalian cathepsin B.
[0006]
[Problems to be solved by the invention]
To date, a cDNA encoding shrimp-derived cathepsin B has not been cloned. There are also few data on the cold-adapted cathepsin B. Therefore, if we can clone the cDNA encoding cathepsin B from the cold-adapted species, the cathepsin B shown above, such as new specificity and high catalytic efficiency, as well as basic knowledge such as the relationship between structure and function. It is thought to contribute to the development of enzymes with different properties. Furthermore, the present invention may be extremely important in biotechnology, and can provide a very useful enzyme when applied to food modifiers, detergents, cosmetic ingredients, pharmaceuticals, etc. It becomes.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
As a result of seeking out cathepsin B derived from a cold-adapted species in nature and conducting extensive screening, the present inventors have succeeded in finding a gene encoding cathepsin B from the liver pancreas of Japanese pink shrimp.
[0008]
Pink tiger shrimp is a cold-adapted species that is distributed in the North Pacific Ocean and North Atlantic Ocean, and usually lives in a low-temperature environment of -1.6 to 5 ° C. Several reports have shown that cold-adapted species of enzymes show substantially higher catalytic efficiencies than their corresponding mammalian enzymes. For example, it has been reported that trypsin, a serine protease particularly well studied among proteases, has 40 times higher catalytic efficiency of salmon trypsin than bovine trypsin.
[0009]
As a result of various studies using genetic engineering techniques, the present inventors have succeeded in cloning a gene encoding cathepsin B from a cold-adapted species of pink shrimp, and the nucleotide sequence and deduced amino acid sequence of the gene are All of this has been clarified and the present invention has been completed. The protein encoded by this gene was named pink shrimp cathepsin B.
[0010]
By incorporating a gene encoding pink shrimp cathepsin B into an appropriate expression vector and transforming an appropriate host cell with the expression vector, pink shrimp cathepsin B can be produced by a gene recombination method. As the expression vector, various known vectors can be used. For example, when the host is Escherichia coli, a series of pUR vectors, pATH vectors, pGEX vectors, etc. can be mentioned. When used as a host, vectors such as pXM and pDC201 can be used.
[0011]
For example, in a system using E. coli, the gene of the present invention was inserted into pET39b (Novagen) and pRSET (Invitrogen), introduced into E. coli, and expression could be induced. It was confirmed by SDS-PAGE that a protein having the desired molecular weight was expressed. In addition, in yeast systems, the yeast expression vector pPICZα into which this gene has been inserted is introduced into the host yeast P. pastoris KM71H strain by electroporation and grown in a medium containing a high concentration (2 mg / ml) of zeocin. Possible transformants were selected. When the activity was confirmed by gelatin zymography, a band having a size corresponding to the target enzyme was detected. In addition, the activity could be confirmed using Z-Arg-Arg-MCA (Bachem), which is a specific substrate for cathepsin B.
[0012]
The present invention includes a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of pink shrimp cathepsin B and having cathepsin B-like enzyme activity. The present invention also includes a prepro form of the cathepsin B-like enzyme. Also included are polypeptides that are 80% or more identical to all or part of the amino acid sequence of cathepsin B. Furthermore, the present invention also includes the signal peptide and propeptide of cathepsin B. This propeptide is also useful as an inhibitor of cathepsin B-like enzyme.
[0013]
And this invention also includes DNA which codes this enzyme, its signal peptide, and a pro peptide.
For example, the present invention includes a DNA that hybridizes under stringent conditions with a DNA comprising a complementary strand of the above-mentioned pink shrimp cathepsin B or a prepro thereof and encodes a protein having cathepsin B-like enzyme activity.
[0014]
The stringent conditions include, for example, a Hybond N ⁺ nylon membrane (Amersham Pharmacia) treated with DNA at 120 ° C. for 20 minutes and a church phosphate buffer (0.5M Na ₂ HPO ₄ , 1 mM). EDTA, 7% SDS) for 5 minutes at 65 ° C and prehybridize. Hybridization is carried out at 65 ° C. for 17 hours by adding the probe obtained by the procedure described below in the same buffer. The membrane after hybridization was treated with 2xSSC containing 0.1% SDS (standard saline citrate; 1xSSC 150 mM NaCl, 15 mM sodium citrate, pH 7.0) at room temperature for 20 minutes at 65 ° C in 1xSSC containing 0.1% SDS. Perform two 20 minute washes. Further, wash at 0.1xSSC containing 0.1% SDS at 65 ° C for 20 minutes to complete the cleaning. Exposure to X-ray film is performed at -80 ° C for 24 hours.
[0015]
In preparation of the probe, for example, a cDNA fragment amplified by PCR is labeled with [α ³² P] -dCTP using Takara random primer DNA labeling kit Ver. 2 (Takara). Thereafter, unreacted isotopes are removed using a centrifugal filter (Millipore).
[0016]
Furthermore, the present invention provides a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added to the amino acid sequences of pink shrimp cathepsin B and its prepro form, and having cathepsin B-like enzyme activity. Also included are DNA encoding and DNA encoding a prepro form of a protein having cathepsin B-like enzyme activity.
[0017]
Further, in the present invention, 1 to 100, preferably 1 to 10, more preferably 1 to several bases are deleted, substituted, or substituted for DNA encoding pink shrimp cathepsin B and its prepro form. The added DNA includes DNA encoding a protein having the activity of pink shrimp cathepsin B.
[0018]
Further, it includes DNA having the ability to produce a polypeptide that is 80% or more identical to all or part of the amino acid sequence of pink shrimp cathepsin B. Furthermore, a protein or preprocathepsin having cathepsin B activity having a homology of 80%, preferably 90%, particularly preferably 95% or more with respect to the DNA sequence encoding cathepsin B or preprocathepsin B DNA encoding a protein that functions as B is included.
[0019]
In the present invention, a primer for detecting this gene, for example, a sequence of 15 bases or more in a base sequence encoding pink shrimp cathepsin B, or one or several deletions, substitutions or additions are made in the sequence. Base sequence.
[0020]
【Example】
The examples are illustrative and do not limit the invention.
[Example 1] Cloning of pink shrimp cathepsin B.
Cloning was performed by the RACE method. The outline is shown in FIG.
[0021]
<Anatomy of Pink Shrimp>
We purchased live pink shrimp from the Saikai fishery cooperative in Ishikawa Prefecture, dissected and collected the liver pancreas, muscles and intestines. Each collected tissue was immediately frozen in liquid nitrogen and stored frozen at −80 ° C. until use.
[0022]
<Extraction of total RNA>
Total RNA was extracted from the hepatopancreas, muscle, and intestine of pink shrimp using ISOGEN (Nippon Gene). About 100 μg of total RNA was obtained from 100 mg of tissue.
[0023]
<Primer design>
Degenerate primers P1 and P2 were created with reference to the well-conserved regions FDAREQW and SCGSCWA of cathepsin B obtained from various mammals (Table 1, FIG. 1).
[0024]
[Table 1]

The position indicates the position number of SEQ ID NO: 1. The 3′- and 5′-AUAP primers are the same sequences as the primers included in the 3′- and 5′-RACE kit. N represents A / C / G / T.
[0025]
<Creation of 1st strand cDNA>
From 1 μg of total RNA extracted from the liver pancreas, 1st strand cDNA was prepared using 3′RACE System for rapid amplification of cDNA ends (GIBCO BRL).
[0026]
<3'RACE>
Using about 1 μg of the above 1st strand cDNA as a template, 100 μl of 20 pmol primer (P1 and 3′-AUAP), 20 nmol dNTP, 10 μl of 10 x Ex taq buffer, 1 U of Ex taq DNA polymerase (Takara) PCR was performed with the reaction solution. PCR was performed using a DNA thermal cycler (PCR system 9700, PERKIN ELMER) for 3 minutes at 94 ° C, followed by heat denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 1 minute. The reaction cycle was performed 30 times and the final extension step was extended by 5 minutes and terminated. Furthermore, PCR was performed under the same reaction conditions using the nested primer P2. Amplified products were detected by ethidium bromide staining after separation by 1% agarose gel electrophoresis (Fig. 1A). Next, the amplified product was inserted into the pGEM-T Easy vector, subcloned, and the base sequence on the 3 ′ end side was determined.
[0027]
<5'RACE>
Based on the sequence determined by the 3'RACE method, the primers P3, P4 and P5 of the antisense strand shown in Table 1 were prepared, and 5'RACE System for rapid amplification of cDNA ends, version 2 (GIBCO BRL) was used. Then, 5'RACE method was performed according to the protocol (Fig. 1A). The obtained PCR fragment was subcloned in the same manner as in the 3′RACE method, and the base sequence on the 5 ′ end side was determined.
[0028]
<Full length isolation and sequencing>
In order to amplify the full-length cDNA, primer P6 was prepared from the 5 ′ end side of the base sequence obtained by 5′RACE. A full-length cDNA encoding pink shrimp cathepsin B was isolated from the above-mentioned 1st strand cDNA using primers P6 and 3′-AUAP (FIG. 1B), and the nucleotide sequence was determined. The determined base sequence and deduced amino acid sequence are shown in FIG.
[0029]
The full length consisted of 1,159 bases including the poly A tail and contained a 987 bp translation region from the start codon (ATG) to the stop codon (TGA). Polyadenylation signal AATAAA was present at bases 1110-1115 in the 3 'untranslated region.
[0030]
<Primary structure of pink shrimp cathepsin B>
The pink shrimp cathepsin B gene encoded 328 amino acid residues and contained a putative 15-residue signal peptide, a 60-residue propeptide and a 253-residue mature enzyme region (Figure 2). . The deduced amino acid sequence was subjected to homology search by BLAST search, and as a result, it showed high homology with mammalian cathepsin B.
[0031]
Fig. 3 shows the alignment of pink shrimp cathepsin B together with mammalian cathepsin B and invertebrate cathepsin B-like cysteine proteases that showed high homology with BLASTP. Since the prepro regions are conserved in the secondary structure rather than the primary structure, they are separated from the mature enzyme region (Fig. 3B) and aligned (Fig. 3A) because of the low homology of the primary structure.
[0032]
Cysteine proteases belonging to the papain superfamily are divided into two subgroups based on sequence: cathepsin L-like enzymes with ERFNIN motif in the pro region and cathepsin B-like enzymes without it (Karrer et al. al., 1993). There was no ERFNIN motif in the pink shrimp cathepsin B. In most cysteine proteases, the conserved GNFD motif (Vernet et al., 1995) was also present in pink shrimp cathepsin B (Fig. 3A).
[0033]
Pink shrimp and mature cathepsin B showed the highest homology (53%) with human cathepsin B. Table 2 shows amino acid sequence homology with cathepsin B or cathepsin B-like enzymes of other organisms.
[0034]
[Table 2]

[0035]
Pink shrimp and mature cathepsin B generally contained all of the amino acid residues characteristic of cysteine protease, and amino acid residues characteristic of cathepsin B. That is, the pink shrimp and mature cathepsin B are the catalytic groups Cys29, His199 and Asn219 (numbers indicate the position number of human mature cathepsin B. The same applies hereinafter), Gln23 and Gly27 that form the oxyanion hole, and the substrate It contained Gly73, Gly74 and Trp221 in the vicinity of the site that interacts with the main chain, and Pro2 present in the vicinity of the N-terminus (Fig. 3B). Cathepsin B is characteristic in that it exhibits exopeptidase activity compared to other cysteine proteases belonging to the papain superfamily. An occluding loop (bases 105 to 126) (Musil et al., 1991), which plays an important role in exhibiting this feature, was also present in pink shrimp cathepsin B. In addition, two consecutive histidine residues (His110 and His111) that are present in this loop and that constitute the S2 ′ subsite (Schechter and Berger notation) were conserved (FIG. 3B). In addition, 12 cysteine residues that form 6 disulfide bonds in human cathepsin B were also conserved in pink shrimp cathepsin B (Fig. 3B).
[0036]
<Strain relations of pink shrimp and cathepsin B>
For typical cathepsins belonging to the pink shrimp cathepsin B, papain, and the papain superfamily, a phylogenetic tree of the mature enzyme excluding the prepro region was prepared. The phylogenetic tree was constructed using the PHYLIP package, version 3.5c (Felsenstein, 1993) by the neighborhood join method. Also in the phylogenetic tree, pink shrimp cathepsin B was classified into the cathepsin B lineage group (Fig. 4).
[0037]
<Structural properties of pink shrimp cathepsin B>
The calculated molecular weight of the pink shrimp and mature cathepsin B was 28,108, which agreed well with the molecular weight ranges of cathepsin B and its homologs known so far. Pink shrimp and mature cathepsin B had the highest negative charge of cathepsin B. The theoretical value of the isoelectric point was significantly lower than that of other homologues and was pI 4.19. The hydropathicity calculated by Kyte and Doolittle (1982) was estimated to be -0.487. From the results of charge, isoelectric point, and hydropathicity values, pink shrimp cathepsin B showed typical structural characteristics of cold-adapted aquatic organisms.
[0038]
<Northern blot analysis>
The following operation was performed according to the method of Sambrook (1989) et al. Total RNA (10 μg) of each tissue described above was electrophoresed, transferred to a Hybond-N + membrane, and then hybridized at 65 ° C. overnight. A DNA fragment 369 bases (696-1064 bases) in the 3 ′ end region was amplified by PCR and labeled with [α-32P] dCTP using Random Primer DNA Labeling kit Ver.2.0 (Takara). As a result of Northern blot analysis, an overwhelmingly strong signal was detected in the liver pancreas (FIG. 5).
[0039]
<Construction of expression system>
In the system using Escherichia coli, the gene of the present invention was inserted into pET39b (Novagen), introduced into Escherichia coli BL21 (DE3) strain, and expression could be induced. It was confirmed by SDS-PAGE that the fusion protein with the desired molecular weight was expressed.
[0040]
In the yeast system, the yeast expression vector pPICZ α (Invitrogen) inserted with this gene was introduced into the host yeast P. Pastoris KM71H strain by electroporation, and a medium containing high concentration (2 mg / ml) of zeocin. Transformants capable of growing in the above were selected. A single colony transformant (KM71H strain) was inoculated into 125 ml of GY (glycerol-complex medium, pH <3.0) and cultured with shaking at 30 ° C. for 72 hours. Next, the culture was collected by centrifugation and resuspended in 15 ml of MM (minimal methanol medium, pH <3.0). Methanol addition was repeated approximately every 24 hours, and cultured with shaking at 26 ° C. for 120 hours. Analysis by SDS-PAGE detected a band of a size corresponding to the protein of interest. Next, when the activity was confirmed by gelatin zymography, a band having a size corresponding to the target enzyme was detected. The activity could be confirmed using Z-Arg-Arg-MCA (Bachem), which is a specific substrate for cathepsin B.
[0041]
REFERENCES
Felsenstein, J., 1993.PHYLIP (Phylogeny Inference Package) version 3.5c.Department of Genetics, University of Washington, Seattle.
Karrer, KM, Peiffer, SL, DiTomas, ME, 1993. Two distinct gene subfamilies within the family of cysteine protease genes.Proc. Natl. Acad. Sci. USA. 90, 3063-3067.
Kyte, J., Doolittle, RF, 1982.A simple method for displaying the hydropathic character of a protein.J. Mol. Biol. 157, 105-132.
Musil, D., Zucic, D., Turk, D., Engh, RA, Mayr, I., Huber, R., Popovic, T., Turk, V., Towatari, T., Katunuma, N., et al., 1991.The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.EMBO J. 10, 2321-2330.
Sambrook, J., Fritsch, EF, Maniatis, T., 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor: Cold Spring Harbor laboratory press, New York.
Schechter, I., Berger, A., 1967. On the size of the active site in proteinases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157-162.
Vernet, T., Berti, PJ, de Montigny, C., Musil, R., Tessier, DC, Menard, R., Magny, MC, Storer, AC, Thomas, DY, 1995. Processing of the papain precursor. ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing.J. Biol. Chem. 270, 10838-10846.
[0042]
[Example 2] The cathepsin B enzyme activity of a crude extract of the pink shrimp liver pancreas.
<Preparation of crude extract>
The crude extract was prepared from the hepatopancreas frozen at -80 ° C. The frozen tissue was partially thawed and homogenized with a 50 mM phosphate buffer (pH 6.5) containing 2 volumes of 150 mM NaCl for 5 minutes using a Polytron homogenizer. After 1/3 times the amount of tetrachloromethane was slowly added and stirred, the mixture was centrifuged to degrease. The defatted supernatant was used as a crude extract.
[0043]
<Measurement of cathepsin B activity>
The activity of the crude extract was measured using Z-Arg-Arg-MCA, which is a specific substrate for cathepsin B. The reaction was performed at a final substrate concentration of 100 μM in 100 mM acetate buffer (pH 5.5) containing 2 mM EDTA, 2 mM DTT, 0.05% Triton X-100. The fluorescence intensity of the released AMC was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm. The measurement was repeated 5 times. A standard curve was prepared to quantify the released AMC, and the amount of enzyme that liberates 1 μmol of AMC per minute at 25 ° C. was defined as 1 U. As a result, the crude extract showed high cathepsin B activity of 438.1 ± 9.1 mU. This activity was inhibited by E-64, a specific inhibitor of cysteine protease. PMSF, which is a specific inhibitor of serine protease, had little effect. From the above, it was suggested that cathepsin B is present in protein scales in the poppy liver pancreas.
[0044]
Ellipses: AMC, 7-amino-4-methylcoumarin; DTT, dithiothreitol; E-64, L-trans-epoxysuccinyl-leucyl-agmatine; MCA, 4-methyl-7-coumarylamide; PMSF, phenylmethylsulfonyl fluoride, Z, benzyloxycarbonyl.
[0045]
【The invention's effect】
According to the present invention, a novel cathepsin B derived from pink shrimp is provided. This enzyme can be obtained from the Arctic shrimp hepatopancreas or by introducing a gene encoding this enzyme and culturing transformed host cells, and is usefully used in a wide range of fields such as food, cosmetics, and medicine. be able to.
[0046]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the outline of cloning of a cDNA encoding pink shrimp cathepsin B. (A) A schematic diagram of the positions of primers used for 3′- and 5′-RACE and an electrophoretogram of each PCR product are shown. (B) A diagram schematically showing isolation of full-length cDNA by single PCR and an electrophoretogram of PCR products. M, DNA size marker; 3 ', 3' RACE product; 5 ', 5' RACE product; F, full-length cDNA.
FIG. 2 is a diagram showing the nucleotide sequence and deduced amino acid sequence of pink shrimp cathepsin B. The bold letters in the base sequence indicate the start codon (ATG) and the stop codon (TGA), and the underline indicates the putative polyadenylation signal (AATAAA) and the poly A tail. The regions surrounded by the white rectangle and the gray rectangle of the amino acid sequence indicate a signal peptide and a propeptide, respectively, and the open portions indicate cysteine, histidine and asparagine residues in the active site.
FIG. 3 shows alignment of pink shrimp cathepsin B together with cathepsin B or cathepsin B-like cysteine proteases of other organisms. The ellipsis indicates Pb, pink shrimp; CtB, cathepsin B; Hs, Homo sapiens; Bt, Bos taurus; Sp, Sarcophaga peregrina; Dm, Drosophila melanogaster; Bm, Bombyx mori; The same amino acid residues and similar amino acid residues as the pink shrimp cathepsin B are shown in black and gray, respectively. (A) shows the alignment of the prepro region. (B) shows the alignment of mature enzymes.
The number on the sequence indicates the position number in human cathepsin B.
S1, S2, S3, S1 ′ and S2 ′ represent amino acid residues constituting the binding pocket. Numbers marked with an asterisk * represent the catalytic groups cysteine, histidine, and asparagine.
A black circle indicates a cysteine residue that forms a disulfide bond in human cathepsin B, and a portion surrounded by a square indicates an occluding loop.
FIG. 4 is a diagram in which a rootless phylogenetic tree was created by the neighbor joining method based on the sequences of all cathepsin B (shown in bold) shown in FIG. 3 and the mature enzymes of various cysteine proteases. The abbreviations indicate Ct, cathepsin; Rn, Rattus norvegicus; Lma, Leishmania major; Lme, Leishmania mexicana; PAM, percent accepted mutation. The numerical value at each branch point indicates the value of the bootstrap test.
FIG. 5 shows the results of Northern blot analysis of mRNA encoding cathepsin B in each tissue of the pink shrimp. 10 μg of total RNA was electrophoresed on a 0.9% agarose gel and then blotted on Hybond-N + membrane. Lane 1, muscle; Lane 2, hepatopancreas; Lane 3, intestine. RNA size markers are shown on the left.

Claims (18)

The following protein (a) or (b).
(A) a protein comprising an amino acid sequence from position number 76 onward in SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a) and having cathepsin B-like cysteine protease activity. The protein according to claim 1, which is the following (a) or (b).
(A) a protein comprising an amino acid sequence from position number 76 onwards of SEQ ID NO: 2,
(B) A protein comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a) and having cathepsin B-like cysteine protease activity.   DNA encoding the protein according to claim 1 or 2. DNA of the following (a) or (b).
(A) DNA comprising 253 to 1011 bases of SEQ ID NO: 1,
(B) A DNA that hybridizes with a DNA containing a complementary base sequence of (a) under a stringent condition and encodes a protein having cathepsin B-like enzyme activity. The DNA according to claim 4, which is the following (a) or (b).
(A) DNA consisting of 253 to 1011 bases of SEQ ID NO: 1,
(B) A DNA that hybridizes with a DNA comprising the complementary base sequence of (a) under a stringent condition and encodes a protein having cathepsin B-like enzyme activity. The following prepro-type cathepsin B-like cysteine protease protein (a) or (b):
(A) a protein consisting of SEQ ID NO: 2,
(B) A prepro form of a protein comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a) and having cathepsin B-like cysteine protease activity.   DNA encoding the prepro-type cathepsin B-like cysteine protease according to claim 6. DNA of the following (a) or (b).
(A) DNA set forth in SEQ ID NO: 1,
(B) A DNA that hybridizes with a DNA comprising a complementary base sequence of (a) under stringent conditions and encodes a prepro form of a protein having cathepsin B-like enzyme activity.   A vector comprising the DNA according to any one of claims 3 to 5 or claims 7 to 8.   A host cell capable of expressing a cathepsin B-like cysteine protease transformed with the vector of claim 9.   A method for producing a cathepsin B-like cysteine protease protein, comprising expressing and recovering a cathepsin B-like protein using the host cell according to claim 10. Signal peptide of cathepsin B-like cysteine protease of the following (a) or (b).
(A) A peptide comprising the amino acid sequence described in position numbers 1 to 15 (Met to Ala) of SEQ ID NO: 2.
(B) A peptide consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a) and functioning as a signal peptide of a cathepsin B-like cysteine protease.   A DNA encoding the signal peptide of the tepsin B-like cysteine protease according to claim 12. DNA of the following (a) or (b).
(A) DNA consisting of 28 to 72 bases of SEQ ID NO: 1,
(B) A DNA that hybridizes with a DNA comprising the complementary base sequence of (a) under stringent conditions and encodes a signal peptide of a cathepsin B-like enzyme. The following (a) or (b) cathepsin B-like cysteine protease propeptide.
(A) A peptide comprising the amino acid sequence described in position numbers 16 to 75 (Glu to Glu) of SEQ ID NO: 2.
(B) A peptide comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a), and capable of functioning as a propeptide of cathepsin B-like cysteine protease.   DNA encoding the propeptide of the cathepsin B-like cysteine protease according to claim 15. DNA of the following (a) or (b).
(A) DNA consisting of 73 to 252 bases of SEQ ID NO: 1,
(B) A DNA that hybridizes with a DNA comprising the complementary base sequence of (a) under stringent conditions and encodes a cathepsin B-like enzyme propeptide.   A primer for detecting a cathepsin B-like cysteine protease gene consisting of 15 or more consecutive DNAs or complementary strands thereof according to any one of claims 3 to 5 or 7 to 8.

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