JP2581823B2 - Human Inflammatory Locally Induced Phospholipase A <2> Inhibitory Protein, Production Method and Gene Thereof - Google Patents
Human Inflammatory Locally Induced Phospholipase A <2> Inhibitory Protein, Production Method and Gene ThereofInfo
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- JP2581823B2 JP2581823B2 JP2089085A JP8908590A JP2581823B2 JP 2581823 B2 JP2581823 B2 JP 2581823B2 JP 2089085 A JP2089085 A JP 2089085A JP 8908590 A JP8908590 A JP 8908590A JP 2581823 B2 JP2581823 B2 JP 2581823B2
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- Prior art keywords
- phospholipase
- human
- inhibitory
- protein
- gene
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Classifications
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- 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
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Description
【発明の詳細な説明】 ＜産業上の利用分野＞ 本発明は炎症性疾患の治療に有用であることが期待さ
れる蛋白に関する。さらに詳しくはヒト炎症局所由来の
ホスホリパーゼA2を阻害するヒト由来の蛋白、その製造
方法及びその遺伝子に関する。The present invention relates to a protein expected to be useful for treating inflammatory diseases. More particularly proteins of human origin for inhibiting phospholipase A 2 derived from human inflammatory sites, a method and to a gene that production.
＜従来の技術＞ ステロイド剤は強力な抗炎症薬として、多くの炎症性
疾患の治療に用いられてきた。しかし、ステロイドはそ
のホルモン作用により様々な副作用を持つため、その使
用は比較的重篤な患者に限らざるを得ない。したがって
ステロイドと同等の抗炎症作用を持ち、かつ副作用の少
ない新規医薬品の開発が、炎症性疾患の治療にあたる医
師にとって最大の要求となっている。<Conventional Technology> Steroids have been used as potent anti-inflammatory drugs for the treatment of many inflammatory diseases. However, steroids have various side effects due to their hormonal effects, and their use must be limited to relatively serious patients. Therefore, the development of new drugs having the same anti-inflammatory action as steroids and having few side effects has become the greatest demand for doctors who treat inflammatory diseases.
ステロイドが抗炎症作用を発揮するメカニズムの一つ
として、炎症局所においてホスホリパーゼA2を阻害する
蛋白を合成誘導することにより、この酵素によるアラキ
ドン酸の遊離を抑制し、その結果起炎的な作用を持つア
ラキドン酸代謝物の産生を低下させることが考えられて
いる（Flower et al.Nature 278,456,1979）。このホス
ホリパーゼA2阻害蛋白は、上記の要求を満足する活性を
持つことが期待されるため、数多くの研究者により、そ
の単離・精製と抗炎症作用評価の試みが行われてきた。One mechanism which steroids exert anti-inflammatory effects, by combining inducing protein that inhibits phospholipase A 2 at inflammatory sites, suppress the release of arachidonic acid by the enzyme, the results pathogenic effects It is thought to reduce the production of arachidonic acid metabolites (Flower et al. Nature 278, 456, 1979). The phospholipase A 2 inhibitory proteins, because it is expected to have the activity to satisfy the above requirements, the number of researchers, its isolation and purification and attempts antiinflammatory effect evaluation has been carried out.
しかしながら、これまでのところステロイドに匹敵す
るほどの強力な抗炎症作用を示すものは見いだされてい
ない。その原因として次の２つが考えられる。However, to date, none have been found to exhibit a potent anti-inflammatory effect comparable to steroids. The following two are considered as the causes.
（１）従来の研究では阻害活性の評価系に膵臓由来の酵
素を用いていたが、炎症局所由来のホスホリパーゼA2は
膵由来ホスホリパーゼA2とは異る構造及び酵素活性を持
つものであること。(1) In the conventional study had using enzymes from the pancreas to the evaluation system of the inhibitory activity, phospholipase A 2 derived from inflammatory sites are those having yl structure and enzymatic activity and pancreatic-derived phospholipase A 2 .
（２）ホスホリパーゼA2に直接作用するものではなく、
基質側との相互作用による見かけ上の阻害活性を検出し
ていたこと。(2) phospholipase A 2 and not act directly on,
Detecting apparent inhibitory activity due to interaction with the substrate side.
本発明者らは、これらの問題点に着目し、まず、カゼ
イン投与により腹膜炎を起こしたラットの腹腔浸出液よ
りホスホリパーゼA2を精製した。そして、この酵素を特
異的に阻害する蛋白を、デキサメサゾン投与ラットの腹
腔洗浄液中に見出し、その精製とＮ末端アミノ酸配列の
決定に成功した（特開昭63−246397号公報）。この阻害
蛋白は膵由来のホスホリパーゼA2に対しては全く阻害活
性を示さなかった。すなわち、この阻害蛋白は、従来の
研究において問題となっていた上記の２点を克服した活
性を持つ点で全くユニークなものであり、従来のホスホ
リパーゼA2阻害蛋白よりはるかに強力な抗炎症作用を示
すことが期待できる。The present inventors focused on these problems, first, purified from peritoneal exudate of rats caused peritonitis phospholipase A 2 casein administration. Then, a protein that specifically inhibits this enzyme was found in peritoneal washings of rats administered with dexamethasone, and its purification and N-terminal amino acid sequence were successfully determined (JP-A-63-246397). This inhibitory protein did not show any inhibitory activity against phospholipase A 2 of膵由come. That is, the inhibitory protein is intended quite unique in having activity which overcomes the above two points, which has been a problem in previous studies, a much stronger anti-inflammatory effect than the conventional phospholipase A 2 inhibitory proteins Can be expected.
この阻害蛋白のＮ末端アミノ酸配列はヒトC3の鎖のア
ミノ酸配列の一部と極めて高いホモロジーを示した。し
たがって、この阻害蛋白はC3の分解産物であると考えら
れた。C3は血清中のプロテアーゼによって段階的に分解
され、C3a,C3b等の様々なフラグメントが生成すること
が知られている。本発明者らがラット腹腔洗浄液より精
製した炎症局所由来ホスホリパーゼA2阻害蛋白はヒトC3
の最終分解産物であるC3dg又はC3dと類似したものであ
ると考えられた（Suwa et al.,Pro.Natl.Acad.Sci.USA,
in press,1990）。その理由は、この阻害蛋白のＮ末端
アミノ酸配列と対応するヒトC3の部位がC3dgまたはC3d
のＮ末端の部位と極めて近似しており、また、分子量が
37kDa又は33kDaであることにある。The N-terminal amino acid sequence of this inhibitory protein showed extremely high homology with a part of the amino acid sequence of the human C3 chain. Therefore, this inhibitory protein was considered to be a degradation product of C3. It is known that C3 is degraded stepwise by proteases in serum, and various fragments such as C3a and C3b are generated. Inflammatory sites derived phospholipase A 2 inhibitory protein which the inventors have purified from rat peritoneal washings human C3
Was considered to be similar to C3dg or C3d, which is the final degradation product of (Suwa et al., Pro. Natl. Acad. Sci. USA,
in press, 1990). The reason is that the site of human C3 corresponding to the N-terminal amino acid sequence of this inhibitory protein is C3dg or C3d
Is very similar to the N-terminal site of
37 kDa or 33 kDa.
そこで本発明者らは、ラット血清よりC3dgを別途精製
し、ラットC3dgが炎症局所由来ホスホリパーゼA2を特異
的に阻害する活性を有することを見出した。また、遺伝
子クローニングにより、ラットC3dgをコードする遺伝子
の全塩基配列及び全アミノ酸配列を決定した（特願平１
−200246）。The present inventors have separately purified C3dg from rat serum, rat C3dg were found to have specifically inhibit activity of inflammation localized derived phospholipase A 2. In addition, the entire nucleotide sequence and the entire amino acid sequence of the gene encoding rat C3dg were determined by gene cloning (Japanese Patent Application No. Hei.
-200246).
＜発明が解決しようとする課題＞ しかしながら、ラット由来のホスホリパーゼA2阻害蛋
白は、抗炎症薬として臨床的な評価を行うのには抗原性
等の点で問題がある。<SUMMARY invention> However, phospholipase A 2 inhibitory proteins derived from rat, to perform clinical evaluation as anti-inflammatory agents have problems in terms of antigenicity and the like.
本発明は、ヒト炎症局所由来ホスホリパーゼA2を阻害
するヒト由来の蛋白の構造を決定すると同時にこの蛋白
の製造法およびこの蛋白を製造するために用いることの
できるその蛋白遺伝子を提供し、抗炎症薬としての開発
を可能ならしめることを目的とする。The present invention provides the protein gene which can be used human inflammatory sites derived phospholipase A 2 This protein preparation at the same time to determine the structure of a protein from human to inhibit and to produce the protein, anti-inflammatory The aim is to make development as a drug possible.
＜課題を解決するための手段＞ すなわち本発明は、第１図に示すアミノ酸配列を有す
るヒト炎症局所由来ホスホリパーゼA2阻害蛋白である。That is, the present invention <Means for Solving the Problems> is a human inflammatory sites derived phospholipase A 2 inhibitory proteins having the amino acid sequence shown in Figure 1.
また本発明は下記工程、 ヒト血清を得て、 該ヒト血清を37℃で６〜10日間処理し、 該処理したヒト血清にプロテア−ゼインヒビターを添
加し、 該プロテア−ゼインヒビターを添加したヒト血清から
該蛋白を精製する からなる第１図に示すアミノ酸配列を有するヒト炎症局
所由来ホスホリパーゼA2阻害蛋白の製造方法である。The present invention also provides the following steps: obtaining human serum, treating the human serum at 37 ° C. for 6 to 10 days, adding a protease inhibitor to the treated human serum, and adding the protease inhibitor to the human serum. a method for producing a human inflammatory sites derived phospholipase a 2 inhibitory proteins having the amino acid sequence shown in Figure 1 consisting of purifying said protein from the serum.
本発明のヒト炎症局所由来ホスホリパーゼA2阻害蛋白
は、第１図に示す349個のアミノ酸配列からなるポリペ
プチドであるが、それと実質的に同等の生物活性が保持
されているならば、上記アミノ酸配列に部分的な置換，
欠失，挿入などがなされて構成されるポリペプチドも本
発明のヒト炎症局所由来ホスホリパーゼA2阻害蛋白に含
まれる。Human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention is a polypeptide consisting of 349 amino acid sequence shown in Figure 1, therewith if substantially equivalent biological activity is retained, the amino acid Partial substitution into an array,
Deletion, the polypeptide constituted by inserting and made are also included in human inflammatory topical derived phospholipase A 2 inhibitory proteins of the present invention.
本発明の製造方法においては、ヒトの血清を酵素処理
して上記アミノ酸配列を有する起炎性ホスホリパーゼA2
阻害蛋白を製造できるが、かかる製造方法における酵素
処理とは、例えば、ヒト血清を直接37℃で６日〜10日間
処理するか、あるいは同血清からC3を精製しこれをFact
or Iを用いて通常の酵素処理操作に付すことをいう。In the production method of the present invention, human serum is subjected to an enzymatic treatment, and the proinflammatory phospholipase A 2 having the above amino acid sequence is used.
An inhibitory protein can be produced. Enzymatic treatment in such a production method includes, for example, treating human serum directly at 37 ° C. for 6 to 10 days, or purifying C3 from the serum to obtain Fact.
It means that it is subjected to a normal enzyme treatment operation using or I.
更にまた、本発明のヒト炎症局所由来ホスホリパーゼ
A2阻害蛋白の遺伝子の一例は第２図に示す1047個のDNA
塩基配列からなるが、一本鎖DNA、又はこの一本鎖DNAと
相補DNAとからなる二本鎖DNAのいずれもその範疇に含ま
れる。さらに上記阻害蛋白のアミノ酸配列をコードする
ものであれば、上記塩基配列に部分的な置換，欠失，挿
入がなされて構成されるDNA、上記塩基配列を含むDNA、
及び上記塩基配列の一部分のみを有するDNAも当然本発
明のヒト炎症局所由来ホスホリパーゼA2阻害蛋白の遺伝
子に含まれる。Furthermore, the phospholipase derived from human inflammation localization of the present invention
An example of a gene of A 2 inhibitory proteins 1047 pieces of DNA shown in Figure 2
Although consisting of a base sequence, both single-stranded DNA and double-stranded DNA consisting of this single-stranded DNA and complementary DNA are included in the category. Furthermore, as long as it encodes the amino acid sequence of the inhibitory protein, a DNA constituted by partially substituting, deleting, or inserting the base sequence, a DNA containing the base sequence,
And included in the gene for human inflammatory sites derived phospholipase A 2 inhibitory protein of a DNA is also naturally present invention having only a portion of the nucleotide sequence.
次に本発明のヒト炎症局所由来ホスホリパーゼA2阻害
蛋白の全アミノ酸配列及びこの阻害蛋白の遺伝子に至る
迄の本発明の手法につき説明する。Next will be described the method of the present invention up to the full amino acid sequence and gene of this inhibitory protein of human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention.
先述したように、本発明者らはラットC3dgがラット炎
症局所由来ホスホリパーゼA2を特異的に阻害する活性を
有することを見出した。As previously described, the present inventors have found that having an activity rat C3dg to specifically inhibit rat inflammatory sites derived phospholipase A 2.
また、本発明者らはヒト炎症局所、すなわち、慢性関
節リウマチ患者関節液よりホスホリパーゼA2を精製する
ことに成功し（特願昭62−325255号、Hara et al,J.Bio
chem.104,326−328 1988）、ヒト炎症局所由来ホスホリ
パーゼA2はラット炎症局所由来ホスホリパーゼA2と構造
及び酵素活性において極めてよく似たものであることを
明らかにした。すなわち、酵素活性においては、両酵素
ともにホスファチジルエタノールアミンに対して高い特
異性を有していた。膵臓由来のホスホリパーゼA2はこの
ような基質特異性を有さない。また、構造については、
Ｎ末端34個のアミノ酸配列を比較した結果、ラット炎症
局所由来ホスホリパーゼA2とヒト炎症局所由来ホスホリ
パーゼA2とのホモロジーは67％であるのに対し、ヒト炎
症局所由来ホスホリパーゼA2とヒト膵臓ホスホリパーゼ
A2とのホモロジーは45％にすぎないことが明らかになっ
た。Further, the present inventors have human inflammatory sites, i.e., succeeded in purifying a phospholipase A 2 from rheumatoid arthritis patients synovial fluid (Japanese Patent Application No. Sho 62-325255, Hara et al, J.Bio
chem.104,326-328 1988), revealed that human inflammatory sites derived phospholipase A 2 are those very similar in rat inflammatory sites derived phospholipase A 2 and the structural and enzymatic activity. That is, both enzymes had high specificity for phosphatidylethanolamine in enzyme activity. Phospholipase A 2 derived from the pancreas do not have such substrate specificity. For the structure,
Result of comparing the N-terminal 34 amino acid sequence, homology with rat inflammatory sites derived phospholipase A 2 with human inflammatory sites derived phospholipase A 2 whereas 67%, human inflammatory sites derived phospholipase A 2 with human pancreatic phospholipase
Homology with A 2 was found to be only 45%.
一方、慢性関節リウマチ患者関節液において、免疫化
学的方法により、高いC3dg値が検出されること、及びそ
の値が病態の重症度と相関を示すことがすでに報告され
ていた（Nydegger et al.J.Clin.Invest.59,862−868 1
977）。On the other hand, it has already been reported that high C3dg levels are detected by immunochemical methods in synovial fluid of patients with rheumatoid arthritis and that the levels show a correlation with the severity of the disease state (Nydegger et al. J. .Clin. Invest. 59,862-868 1
977).
以上のような知見から、本発明者らはヒトにおいても
C3dgが炎症局所由来のホスホリパーゼA2を特異的に阻害
する可能性が大であることをいち早く察知し、この点に
ついて鋭意検討した結果、本発明に到達したのである。From the above findings, the present inventors have found that
C3dg will quickly perceive that potential to specifically inhibit phospholipase A 2 derived from inflammatory sites is large, a result of intensive studies on this point, it has been reached to the present invention.
本発明のヒト炎症局所由来ホスホリパーゼA2阻害蛋白
は、ヒト血清を37℃処理した後アニオン交換HPLC、ゲル
過HPLC、逆相HPLC等によって単離・精製されるか、又
はヒト血清より精製されたC3を酵素処理後、ゲル過HP
LC、逆相HPLC等によって単離・精製される。The human inflammatory local origin phospholipase A 2 inhibitory protein of the present invention is isolated and purified by anion exchange HPLC, gel permeation HPLC, reverse phase HPLC, etc. after treating human serum at 37 ° C., or purified from human serum. After enzyme treatment of C3, gel over HP
It is isolated and purified by LC, reverse phase HPLC, etc.
また、本発明のヒト炎症局所由来ホスホリパーゼA2阻
害蛋白は、C3の分解産物であることから、本蛋白をコー
ドする遺伝子は、すでに報告されているヒトC3遺伝子
（de Bruijn et al.Proc.Natl.Acad.Sci.USA,82,708−7
12,1985）の一部であると考えられた。そこで前述のご
とく精製された本発明のヒト炎症局所由来ホスホリパー
ゼA2阻害蛋白のＮ末端アミノ酸配列を決定し、これを指
標として本阻害蛋白の遺伝子の全塩基配列を決定した。Also, human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention, since it is a degradation product of C3, the gene encoding this protein, human C3 that has already been reported gene (de Bruijn et al.Proc.Natl .Acad.Sci.USA, 82,708-7
12,1985). Therefore to determine the N-terminal amino acid sequence of human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention previously described as purification, to determine the entire base sequence of the present inhibiting protein gene as an index.
さらに、前述のごとく決定した本阻害蛋白遺伝子の全
塩基配列より、本阻害蛋白の全アミノ酸配列を決定し
た。Further, the entire amino acid sequence of the present inhibitory protein was determined from the entire nucleotide sequence of the present inhibitory protein gene determined as described above.
本発明において用いられた各種試験，測定法は以下の
通りである。Various tests and measuring methods used in the present invention are as follows.
（１）ヒト炎症局所由来ホスホリパーゼA2阻害活性の測
定法。(1) Measurement of human inflammatory sites derived phospholipase A 2 inhibitory activity.
酵素として慢性関節リウマチ患者関節液より単離・精
製したホスホリパーゼA2を用い、基質としては14C−酢
酸と共に培養したE.Coliより抽出，精製した14C−ホス
ファチジルエタノールアミン（約2,000dpm/mmol.1mM）
を用いた。Rheumatoid arthritis patients synovial fluid phospholipase A 2 was isolated and purified from used as the enzyme, as the substrate extracted from E.Coli cultured with 14 C-acetate, purified 14 C-phosphatidylethanolamine (about 2,000dpm / mmol .1mM)
Was used.
酵素反応は、50μの0.5M Tris・Cl（pH9.0）,25μ
の40mM CaCl2,25μの基質にサンプル及び水を加え
て総量240μとして混合し最後に10μの酵素液（0.1
ng/μ）を加え、37℃にて10分間反応させたのち、Dol
eの試薬（1.5ml）を加えて反応を停止し、Doleの方法に
従って生成した14C−脂肪酸を抽出し、液体シンチレー
ション・カウンターで測定した。Enzyme reaction was performed using 50μ of 0.5M Tris · Cl (pH 9.0), 25μ
Of 40 mM CaCl 2 , 25 μl of the substrate and the sample and water were added to make a total volume of 240 μl. Finally, 10 μl of the enzyme solution (0.1
ng / μ), and allowed to react at 37 ° C for 10 minutes.
The reaction was stopped by adding the reagent (e) (1.5 ml), and the 14 C-fatty acid produced according to the method of Dole was extracted and measured with a liquid scintillation counter.
（２）SDS−ポリアクリルアミド電気泳動およびウェス
タン・ブロッティング 酵素処理またはHPLCにより得られたサンプルに1/10量
の色素液（0.1％BPB,XC,10％SDS）を加え、100℃５分間
熱処理後、12.5％ポリアクリルアミドゲルにアブライし
１％SDS存在下、15−25mAで1.5時間電気泳動した。還元
状態での解析のためにはサンプルに２−メルカプトエタ
ノール（2ME）を加えた。泳動後、蛋白の検出のためにC
BBで染色した。(2) SDS-polyacrylamide gel electrophoresis and Western blotting 1/10 amount of dye solution (0.1% BPB, XC, 10% SDS) was added to the sample obtained by enzyme treatment or HPLC, and after heat treatment at 100 ° C for 5 minutes And ablated on a 12.5% polyacrylamide gel, and electrophoresed at 15-25 mA for 1.5 hours in the presence of 1% SDS. For analysis in the reduced state, 2-mercaptoethanol (2ME) was added to the sample. After electrophoresis, C
Stained with BB.
また、同様に泳動した後ミリポア社エレクトロブロッ
ティング装置を用いて、蛋白をゲルからニトロセルロー
ス・フィルターに移した。そして、抗ヒトC3dヒツジ血
清（Dako），西洋・ワサビペルオキシダーゼ結合抗ヒツ
ジIgG抗体（Cappel）および基質として４−クロロ−１
−ナフトール（Bio−Rad）を用い、酵素抗体染色法によ
りC3dのバンドを検出した。After electrophoresis in the same manner, the protein was transferred from the gel to a nitrocellulose filter using a Millipore electroblotting apparatus. Then, anti-human C3d sheep serum (Dako), horseradish peroxidase-conjugated anti-sheep IgG antibody (Cappel) and 4-chloro-1 as a substrate
-C3d band was detected by an enzyme antibody staining method using naphthol (Bio-Rad).
（３）蛋白Ｎ末端アミノ酸配列の決定法 気相プロテイン・シークエンサー（アプライド・バイ
オシステム477A）およびHPLC（アプライド・バイオシス
テム120A）を用いた。(3) Method for determining N-terminal amino acid sequence of protein A gas-phase protein sequencer (Applied Biosystem 477A) and HPLC (Applied Biosystem 120A) were used.
即ち、0.1％TFAに溶解したサンプル100μを、上記
気相プロテインシークエンサーにアプライした。自動的
にエドマン分解されたPTH（フェニルチオヒダントイ
ン）アミノ酸誘導体は、その後、高速液体クロマトグラ
フィーにて各アミノ酸として分析された。That is, 100 μm of a sample dissolved in 0.1% TFA was applied to the above-mentioned gas-phase protein sequencer. The PTH (phenylthiohydantoin) amino acid derivative that was automatically Edman-degraded was then analyzed as each amino acid by high performance liquid chromatography.
本発明のヒト炎症局所由来ホスホリパーゼA2阻害蛋白
は、本発明によってその全アミノ酸配列が明らかになっ
たので、公知の化学合成法によって製造することもでき
るが、遺伝子組換え法によって容易かつ大量に製造する
ことができる。Human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention, since the entire amino acid sequence has been revealed by the present invention, it can also be produced by known chemical synthesis methods, easily and in large quantities by genetic recombination Can be manufactured.
＜実施例＞ 以下実施例により本発明を更に詳細に説明する。<Example> Hereinafter, the present invention will be described in more detail with reference to examples.
実施例１ ヒト炎症局所由来ホスホリパーゼA2阻害蛋白（以下、
「阻害蛋白」と略す）の調製及び活性測定 （Ａ）阻害蛋白の調製 （１）ヒト血清30mlに0.1％NaN3を加え、37℃で10日間
処理した。Example 1 Human inflammatory local origin phospholipase A 2 inhibitory protein (hereinafter, referred to as
Preparation of Inhibitory Protein) and Measurement of Activity (A) Preparation of Inhibitory Protein (1) To 30 ml of human serum, 0.1% NaN 3 was added and treated at 37 ° C. for 10 days.
処理及び未処理サンプルをそれぞれ50倍希釈後、常法
にしたがってSDS−PAGE後ウェスタン・ブロッティング
によりフィルターに移した。このフィルターについて抗
ヒトC3d血清を用いて酵素抗体染色法を行った結果、未
処理血清では100kDa以上のバンドのみが検出され、37℃
処理血清では39kDaのバンドのみが検出された（図
３）。この結果から、37℃処理により、C3が血清中の酵
素によって切断されてC3dgが生成していることが確認さ
れた。The treated and untreated samples were each diluted 50-fold, then transferred to a filter by SDS-PAGE and Western blotting according to a conventional method. As a result of performing an enzyme-antibody staining method using anti-human C3d serum on this filter, only a band of 100 kDa or more was detected in the untreated serum, and 37 ° C.
Only a band of 39 kDa was detected in the treated serum (FIG. 3). From this result, it was confirmed that C3 was cleaved by the enzyme in the serum to generate C3dg by the treatment at 37 ° C.
そこでこの血清にプロテアーゼ・インヒビター（20μ
g/mlアプロチニン、10μg/mlダイズトリプシンインヒビ
ター、0.5mM EDTA）を加えた後、３の20mM Tris・ＨC
l,pH7.5に対して２回透析を行った。Therefore, a protease inhibitor (20 μl) was added to this serum.
g / ml aprotinin, 10 μg / ml soybean trypsin inhibitor, 0.5 mM EDTA), and then 3 20 mM Tris · HC
Dialysis was performed twice against pH 7.5.
（２）（１）で得られた透析後のサンプル（蛋白濃度約
30mg/ml）を20倍希釈し、遠心分離操作により不溶物を
取り除いた後、以下のようにして目的蛋白を単離・精製
した。(2) The dialyzed sample obtained in (1) (with a protein concentration of about
(30 mg / ml) was diluted 20-fold, and insoluble matter was removed by centrifugation. The target protein was isolated and purified as follows.
まず、サンプルを分取用サイズのアニオン交換HPLC
（TSKgelDEAE−5PW）で分画した（第４図）。第４図ａ
において、実線はUV280nmにおける吸収、破線はNaCl濃
度勾配を示す。溶出は20mM Tris−ＨCl,pH7.5−NaCl
（Ｏ→0.35M）,2.5ml/min,2min/tubeで行った。カラム
サイズは21.5mmφ×30cmである。First, the sample was subjected to preparative size anion exchange HPLC.
(TSKgelDEAE-5PW) (FIG. 4). FIG. 4a
In, the solid line indicates the absorption at UV 280 nm, and the broken line indicates the NaCl concentration gradient. Elution is 20 mM Tris-HCl, pH 7.5-NaCl
(O → 0.35M), 2.5ml / min, 2min / tube. The column size is 21.5mmφ × 30cm.
次いで、第４図ｂに示されたフラクション中のSDS−P
AGEで39kDaのバンドが検出されたフラクションNr.52−5
4の部分を集め４倍希釈後、これを分析用サイズのアニ
オン交換HPLC（TSKgelDEAE−5PW）で精製した（第５
図）。第５図ａにおいて実線はUV280nmにおける吸収、
破線はNaCl濃度勾配を、棒グラフはヒト炎症局所由来ホ
スホリパーゼA2阻害活性を示す。Then, SDS-P in the fraction shown in FIG.
Fraction Nr. 52-5 in which a band of 39 kDa was detected in AGE
The part 4 was collected and diluted 4-fold, and then purified by analytical size anion exchange HPLC (TSKgelDEAE-5PW) (No. 5).
Figure). In FIG. 5a, the solid line is the absorption at UV 280 nm,
A broken line indicates a NaCl gradient, the bar graph shows the human inflammatory sites derived phospholipase A 2 inhibitory activity.
精製は、20mM Tris−ＨCl,pH7.5−NaCl（Ｏ→1.0M）,
1.0ml/min,2min/tubeで行った。Purification was performed using 20 mM Tris-HCl, pH 7.5-NaCl (O → 1.0 M),
The test was performed at 1.0 ml / min and 2 min / tube.
次に第５図ｂ中のSDS−PAGEで39kDaのバンドが検出さ
れ、ヒト炎症局所由来ホスホリパーゼA2阻害活性の最も
強かったフラクションNr.13のピーク部分を集め、ゲル
過HPLC（TSKgelG3000SW）で更に分画した（第６
図）。第６図ａにおいて、実線はUV 280nmにおける吸
収を、棒グラフはヒト炎症局所由来ホスホリパーゼA2阻
害活性を示す。Then the band of 39kDa was detected by SDS-PAGE of the 5 in the figure b, collected strongest was the peak portion of the fraction Nr.13 human inflammatory sites derived phospholipase A 2 inhibitory activity, further by gel over HPLC (TSKgelG3000SW) Fractionated (6th
Figure). In Figure 6 a, the solid line the absorbance at UV 280 nm, bar graph shows the human inflammatory sites derived phospholipase A 2 inhibitory activity.
溶出条件は20mM Tris−ＨCl,pH7.5−1M NaCl 0.5ml/
min,2min/tubeで行った。Elution conditions were 20 mM Tris-HCl, pH 7.5-1 M NaCl 0.5 ml /
min, 2 min / tube.
最後に第６図ｂ中のSDS−PAGEで39kDaのバンドが検出
され、ヒト炎症局所由来ホスホリパーゼA2阻害活性の最
も強かったフラクションNr.20,21を更に逆相HPLC（Bio
−Rad RP304）で精製した（第７図）。第７図ａにおい
て実線はUV210nmにおける吸収、破線はCH3CN濃度勾配を
示す。精製は、0.1％TFA/CH3CNO〜80％,1ml/min,2min/t
ubeで行った。Finally bands 39kDa by SDS-PAGE in FIG. 6 b is detected, further reverse phase HPLC (Bio the strongest were fractions of human inflammatory sites derived phospholipase A 2 inhibitory activity Nr.20,21
-Rad RP304) (FIG. 7). In FIG. 7a, the solid line shows the absorption at UV 210 nm, and the broken line shows the CH 3 CN concentration gradient. Purification is 0.1% TFA / CH 3 CNO-80%, 1 ml / min, 2 min / t
I went with ube.
第７図ｂ中のSDS−PAGEに示したように分子量39kDaの
阻害蛋白はフラクションNr.30に溶出したが、フラクシ
ョンNr.23に主に溶出しているアルブミンとみられる蛋
白が若干混在していることがわかった。しかし、アルブ
ミンは強いホスホリパーゼA2阻害活性を持たないこと、
及びフラクションNr.23もホスホリパーゼA2阻害活性を
示さなかったことから、阻害蛋白の活性測定には影響を
及ぼさないものと考えた。As shown by SDS-PAGE in FIG. 7b, the inhibitory protein with a molecular weight of 39 kDa eluted in fraction Nr.30, but the fraction Nr.23 contained some proteins that seemed to be mainly eluted with albumin. I understand. However, albumin does not have strong phospholipase A 2 inhibitory activity,
And since the fraction Nr.23 showed any phospholipase A 2 inhibitory activity, the activity measurement of the inhibitory protein was considered to not affect.
（Ｂ）阻害蛋白のホスホリパーゼA2阻害活性 （Ａ）で得られた本発明の阻害蛋白（逆相HPLCフラク
ションNr.30、蛋白濃度約15ng/μ）のヒト炎症局所由
来ホスホリパーゼA2及びその他のホスホリパーゼA2に対
する阻害活性を、前述のホスホリパーゼA2阻害活性の測
定法によって測定した。(B) Inhibition protein of the present invention obtained in inhibitory protein of phospholipase A 2 inhibitory activity (A) (reverse phase HPLC fractions Nr.30, protein concentration of about 15 ng / mu) inflammatory sites derived phospholipase A 2 and other human the inhibitory activity against phospholipase a 2, was measured by the above-mentioned method for the determination of phospholipase a 2 inhibitory activity.
その結果を第８図及び下記第１表に示した。 The results are shown in FIG. 8 and Table 1 below.
第８図から本阻害蛋白はヒト炎症局所由来ホスホリパ
ーゼA2を用量依存的に阻害することが明らかである。ま
た、1ngのヒト炎症局所由来ホスホリパーゼA2活性を阻
害するのに必要な阻害蛋白量は約50ngであった。さらに
第１表から明らかなように、本阻害蛋白はラット炎症局
所由来ホスホリパーゼA2は阻害したが、ブタ膵臓ホスホ
リパーゼA2に対しては有意な阻害活性を示さなかった。 This inhibitory protein from the FIG. 8 it is apparent to inhibit human inflammatory sites derived phospholipase A 2 in a dose-dependent manner. Furthermore, inhibitory protein amount required to inhibit human inflammatory sites derived phospholipase A 2 activity of 1ng was about 50 ng. As further apparent from Table 1, the inhibitory protein is rat inflammatory sites derived phospholipase A 2 is inhibited, it showed no significant inhibitory activity against porcine pancreatic phospholipase A 2.
以上の結果から、本阻害蛋白は炎症局所由来ホスホリ
パーゼA2を特異的に阻害する活性を有することが明らか
である。From the above results, the inhibitory protein is found to have an activity to specifically inhibit inflammation localized derived phospholipase A 2.
実施例２ ヒト炎症局所由来ホスホリパーゼA2阻害蛋白遺伝子の全
塩基配列及び本阻害蛋白の全アミノ酸配列の決定 （Ａ）阻害蛋白の同定 （１）阻害蛋白のＮ末端アミノ酸配列の決定 実施例１（Ａ）で単離・精製した分子量約39kDaの阻
害蛋白（逆相HPLCフラクションNr.30）について、前述
した方法でＮ末端アミノ酸配列を決定した。その結果、
下記のような配列であることが判明した。Example 2 Human inflammatory sites derived phospholipase A 2 inhibitory proteins complete nucleotide sequence and determination of the entire amino acid sequence of the inhibitory protein genes (A) Identification of inhibitory protein (1) Determination Example of N-terminal amino acid sequence of the inhibitory protein 1 ( The N-terminal amino acid sequence of the inhibitory protein having a molecular weight of about 39 kDa (reverse-phase HPLC fraction Nr. 30) isolated and purified in A) was determined by the method described above. as a result,
The following sequence was found.
NH２-Glu-Gly-Val-Asn-Lys-Glu-Asp-Ile-Pro-Pro- これは既に知られているヒトC3dgのＮ末端アミノ酸配
列と完全に一致した。 NH 2 -Glu-Gly-Val- Asn-Lys-Glu-Asp-Ile-Pro-Pro- which was entirely consistent with previously known N-terminal amino acid sequence of human C3dg.
（２）阻害蛋白の免疫化学的解析 実施例１（Ａ）で単離・精製した分子量約39kDaの阻
害蛋白（逆相HPLCフラクションNr.30）について、前述
した方法でSDS−PAGE後、ウェスタン・ブロッティング
を行ってフィルターに移し、抗ヒトC3d血清を用いて酵
素抗体染色法により検出した。その結果、本阻害蛋白は
抗ヒトC3d血清と特異的に反応した。第９図ａにSDS−PA
GEの結果を示す。第９図ｂにウェスタン・ブロッティン
グの結果を示す。(2) Immunochemical analysis of inhibitory protein The inhibitory protein isolated and purified in Example 1 (A) having a molecular weight of about 39 kDa (reverse-phase HPLC fraction Nr. 30) was subjected to SDS-PAGE according to the method described above, followed by Western blot analysis. The cells were blotted, transferred to a filter, and detected by an enzyme-antibody staining method using anti-human C3d serum. As a result, this inhibitory protein reacted specifically with anti-human C3d serum. Fig. 9a shows SDS-PA
The result of GE is shown. FIG. 9b shows the results of Western blotting.
以上、（１），（２）の結果から、本阻害蛋白がヒト
C3dgであることは明らかである。As described above, from the results of (1) and (2), this inhibitory protein is
Obviously it is C3dg.
（Ｂ）ヒト炎症局所由来ホスホリパーゼA2阻害蛋白（C3
dg）遺伝子の全塩基配列と本阻害蛋白の全アミノ酸配列 ヒトC3については既にcDNAクローニングが行われてお
り、その全塩基配列及びそれから推定される全アミノ酸
配列が決定されている。また、C3遺伝子のなかでC3dgを
コードする部分も決定されている（de Bruijn et al.Pr
oc.Natl.Acad.Sci.USA,82,708−712,1985）。(B) human inflammatory sites derived phospholipase A 2 inhibitory proteins (C3
dg) The entire nucleotide sequence of the gene and the entire amino acid sequence of the present inhibitory protein cDNA cloning of human C3 has already been performed, and the entire nucleotide sequence and the entire amino acid sequence deduced therefrom have been determined. In addition, the part encoding C3dg in the C3 gene has been determined (de Bruijn et al. Pr.
oc. Natl. Acad. Sci. USA, 82, 708-712, 1985).
前述の結果から本発明のヒト炎症局所由来ホスホリパ
ーゼA2阻害蛋白はヒトC3dgであることが明らかである。
したがって、本阻害蛋白遺伝子の塩基配列はヒトC3遺伝
子の全塩基配列のうちC3dgをコードする部分と完全に一
致すると考えられる。また、本阻害蛋白の全アミノ酸配
列はヒトC3遺伝子の全塩基配列から推定されるヒトC3の
全アミノ酸配列のうちC3dgに相当する部分と完全に一致
すると考えられる。Human inflammatory sites derived phospholipase A 2 inhibitory proteins of the present invention from the foregoing results it is clear that a human C3dg.
Therefore, it is considered that the nucleotide sequence of the present inhibitor protein gene completely matches the portion encoding C3dg in the entire nucleotide sequence of the human C3 gene. Further, it is considered that the entire amino acid sequence of the present inhibitory protein completely matches the portion corresponding to C3dg in the entire amino acid sequence of human C3 deduced from the entire nucleotide sequence of the human C3 gene.
以上のことから本発明のヒト炎症局所由来ホスホリパ
ーゼA2阻害蛋白の全アミノ酸配列とヒト炎症局所由来ホ
スホリパーゼA2阻害蛋白遺伝子の全塩基配列は第１図及
び第２図のものと判断された。ヒト炎症局所由来ホスホ
リパーゼA2阻害蛋白は1047個の塩基でコードされ、349
個のアミノ酸からなる蛋白であった。More than that human inflammatory sites derived phospholipase A complete amino acid sequence of 2 inhibitory protein and human inflammatory sites derived phospholipase A 2 inhibitory entire nucleotide sequence of the protein gene of the present invention from being determined that the first view and the second view. Human inflammatory sites derived phospholipase A 2 inhibitory proteins are encoded by the 1047 bases, 349
It was a protein consisting of amino acids.
第１図は、本発明のヒト炎症局所由来ホスホリパーゼA2
阻害蛋白のアミノ酸配列を示す。 第２図は、本発明のヒト炎症局所由来ホスホリパーゼA2
阻害蛋白遺伝子のDNA塩基配列の一例を示す。 第３図は、未処理及び37℃処理したヒト血清のウェスタ
ンブロッティングの結果を示す。 第４図は、分取用サイズのアニオン交換HPLCを用いた場
合のヒト炎症局所由来ホスホリパーゼA2阻害蛋白の溶出
パターン（第４図ａ）とSDS−PAGE（同ｂ）を示す。 第５図は、分析用サイズのアニオン交換HPLCを用いた場
合のヒト炎症局所由来起炎性ホスホリパーゼA2阻害蛋白
の溶出パターン（第５図ａ）とSDS−PAGE（同ｂ）を示
す。 第６図は、ゲル過HPLCを用いた場合のヒト炎症局所由
来起炎性ホスホリパーゼA2阻害蛋白の溶出パターン（第
６図ａ）とSDS−PAGE（同ｂ）を示す。 第７図は、逆相HPLCを用いた場合のラット起炎性ホスホ
リパーゼA2阻害蛋白の溶出パターン（第７図ａ）とSDS
−PAGE（同ｂ）を示す。 第８図は、本発明の阻害蛋白の阻害活性を示す。 第９図は、ヒト炎症局所由来起炎性ホスホリパーゼA2阻
害蛋白のSDS−PAGE（第９図ａ）及びウェスタンブロッ
ティング（同ｂ）の結果を示す。FIG. 1 shows the phospholipase A 2 derived from the human inflammation site of the present invention.
2 shows the amino acid sequence of the inhibitory protein. FIG. 2 shows the phospholipase A 2 derived from the human inflammation site of the present invention.
1 shows an example of a DNA base sequence of an inhibitory protein gene. FIG. 3 shows the results of Western blotting of untreated and 37 ° C.-treated human serum. 4 shows a human inflammatory sites derived phospholipase A 2 inhibitory protein elution pattern in the case of using an anion exchange HPLC size prep (Fig. 4 a) and SDS-PAGE (the b). Figure 5 shows human inflammatory sites from an inflammatory phospholipase A 2 inhibitory protein elution pattern in the case of using an anion-exchange HPLC of analytical size (Fig. 5 a) and SDS-PAGE (the b). Figure 6 shows the elution pattern of human inflammatory sites from an inflammatory phospholipase A 2 inhibitory proteins in the case of using the gel over HPLC (Figure 6 a) and SDS-PAGE (the b). Figure 7 is the elution pattern of rat pathogenic phospholipase A 2 inhibitory proteins in the case of using the reverse phase HPLC with (FIG. 7 a) SDS
-PAGE (same as b) is shown. FIG. 8 shows the inhibitory activity of the inhibitory protein of the present invention. Figure 9 shows the results of SDS-PAGE of human inflammatory sites from an inflammatory phospholipase A 2 inhibitory proteins (Fig. 9 a) and Western blotting (the b).
───────────────────────────────────────────────────── フロントページの続き (72)発明者 工藤 一郎 東京都文京区本郷７丁目３番１号 (72)発明者 井上 圭三 東京都文京区本郷７丁目３番１号 (56)参考文献 Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，134［４ ］（1985）Ｐ．2571−2579 Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃ ｉ．ＵＳＡ，82（1985）Ｐ．708−712 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ichiro Kudo 7-3-1 Hongo, Bunkyo-ku, Tokyo (72) Inventor Keizo Inoue 7-3-1 Hongo, Bunkyo-ku, Tokyo (56) References Immunology, 134 [4] (1985) p. 2571-2579 Proc. Natl. Acad. Sc i. USA, 82 (1985) p. 708-712
Claims (2)
症局所由来ホスホリパーゼA2阻害蛋白。1. A human inflammatory topical derived phospholipase A 2 inhibitory proteins having the amino acid sequence shown in Figure 1.
加し、 該プロテア−ゼインヒビターを添加したヒト血清から
該蛋白を精製する からなる請求項１記載のヒト炎症局所由来ホスホリパー
ゼA2阻害蛋白の製造方法。2. The following steps: obtaining human serum; treating said human serum at 37 ° C. for 6 to 10 days; adding a protease inhibitor to said treated human serum; and adding said protease inhibitor. method for producing a human inflammatory sites derived phospholipase a 2 inhibitory protein of claim 1, wherein consisting purifying said protein from human serum.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2089085A JP2581823B2 (en) | 1990-04-05 | 1990-04-05 | Human Inflammatory Locally Induced Phospholipase A <2> Inhibitory Protein, Production Method and Gene Thereof |
| PCT/JP1990/000996 WO1991001999A1 (en) | 1989-08-03 | 1990-08-03 | Phospholipase a2 inhibiting protein originating in inflamed region, production thereof, and gene therefor |
| EP19900911691 EP0436737A4 (en) | 1989-08-03 | 1990-08-03 | Phospholipase a 2? inhibiting protein originating in inflamed region, production thereof, and gene therefor |
| US08/047,379 US5344764A (en) | 1989-08-03 | 1993-04-16 | Protein inhibitors of phospholipase A2 purified from inflammatory sites and production process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2089085A JP2581823B2 (en) | 1990-04-05 | 1990-04-05 | Human Inflammatory Locally Induced Phospholipase A <2> Inhibitory Protein, Production Method and Gene Thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03287600A JPH03287600A (en) | 1991-12-18 |
| JP2581823B2 true JP2581823B2 (en) | 1997-02-12 |
Family
ID=13961031
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|---|---|---|---|
| JP2089085A Expired - Fee Related JP2581823B2 (en) | 1989-08-03 | 1990-04-05 | Human Inflammatory Locally Induced Phospholipase A <2> Inhibitory Protein, Production Method and Gene Thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2581823B2 (en) |
-
1990
- 1990-04-05 JP JP2089085A patent/JP2581823B2/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Ｊ．Ｉｍｍｕｎｏｌｏｇｙ，１３４［４］（１９８５）Ｐ．２５７１−２５７９ |
| Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８２（１９８５）Ｐ．７０８−７１２ |
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