JP2004097134A - Method for synthesizing long-chain fatty acid by chain-elongating enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for elongating chain length of a saturated fatty acid by utilizing enzymatic activity. <P>SOLUTION: The method for synthesizing the long-chain fatty acid comprises a step for preparing a transformant by introducing a chain-elongating enzyme gene into an adequate host and a step for expressing the chain-elongating enzyme by culturing the transformant and converting C18:0 saturated fatty acid to C20:0 saturated fatty acid by chain-elongating reaction specific to a saturated fatty acid mediated by the enzyme. <P>COPYRIGHT: (C)2004,JPO

Description

ＰＣＲプログラム
▲１▼９５℃　２分
▲２▼９５℃　３０秒
▲３▼５５℃　３０秒
▲４▼７２℃　１分　　▲２▼〜▲４▼を３５サイクル
▲５▼７２℃　１０分
このＰＣＲにより増幅された１６５ｂｐの遺伝子断片の配列を決定し、ネステッド　ＰＣＲ用の以下のプライマーを作製した。
【００３９】
配列番号５のｒＢ２０７Ｆ：５’−ＡＣＴＧＴＧＣＴＣＣＴＧＴＡＣＴＣＴＴＧＧＴＡＣＴＣＣＴＡ−３’
配列番号１３のｒＢ２０７Ｒ：５’−ＣＡＴＧＡＡＣＣＡＡＣＣＡＣＣＣＣＣＡＧＣＴＡＣＣＡＴＧＴ−３’
《５’―ＲＡＣＥ　ＰＣＲ》
Ｒａｔ　ｃＤＮＡ　ｌｉｂｒａｒｙ，　ｌｉｖｅｒ　（Ｔａｋａｒａ）は、ｐＡＰｎｅｏ３ベクターにｃＤＮＡが一定方向にクローニングされている。ｐＡＰｎｅｏ３ベクター上の配列から３種類のＰＣＲ用プライマー配列番号１４のＡ１４３（５’−ＣＴＴＡＧＧＡＣＧＣＧＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧ−３’）、　配列番号４のＡ１４５　（５’−ＴＴＡＴＣＡＴＧＴＣＴＧＧＡＴＣＣＣＴＣＧＡＧＧＧＡＴＣＣＧ−３’）、及び、配列番号１５のＡ１４６　（５’−ＴＴＴＴＣＡＣＴＧＣＡＴＴＣＴＡＧＴＴＧＴＧＧＴＴＴＧＴＣＣ−３’）を設計した。このうちＡ１４３は、ベクターにクローニングされているｃＤＮＡの５’上流側の配列に相当しており、５’ＲＡＣＥ　ＰＣＲを行うのに用いた。このＡ１４３プライマーとｒＡＷ４７５ＲＰ１プライマー及びｒＢ２０７Ｒプライマーを用いて種々の条件で５’―ＲＡＣＥ　ＰＣＲを試みたが、目的の遺伝子断片は単離できなかった。
【００４０】
《３’―ＲＡＣＥ　ＰＣＲ》
３’側の未取得部分を得るために、以下の条件で３’―ＲＡＣＥ　ＰＣＲを行った。なお、プライマーＡ１４５はｐＡＰｎｅｏ３ベクターにクローニングされているｃＤＮＡの３’上流側の配列に対応している。
【００４１】

ＰＣＲプログラム
▲１▼９５℃　５分
▲２▼９５℃　１分
▲３▼５５℃　１分
▲４▼７２℃　３分　　▲２▼〜▲４▼を３５サイクル
▲５▼７２℃　１０分
アガロースゲル電気泳動による解析の結果、目的部分と思われるＤＮＡ断片が増幅されたことが確認できたので、ＰＣＲ反応液をテンプレートとしてさらにネステッド　ＰＣＲを行った。
【００４２】

ＰＣＲプログラム
▲１▼９５℃　５分
▲２▼９５℃　１分
▲３▼５５℃　１分
▲４▼７２℃　３分　　▲２▼〜▲４▼を３５サイクル
▲５▼７２℃　１０分
このＰＣＲにより約２３００　ｂｐのＤＮＡ断片が増幅されたのでシークエンス解析を行った結果、目的の断片であることが確認された。
【００４３】
《ｒＥＬＯ２全長遺伝子の単離》
マウスのＥＳＴクローンＡＷ４７５６８１の配列を元に設計した、配列番号６記載の開始コドンを含むプライマーｒａｔＥｌｏＦ１（５’−ＡＴＣＡａａｇｃｔｔＡＴＧＡＡＣＡＴＧＴＣＡＧＴＧＴＧＧＡＣＴＴＴＡＣ−３’；小文字部分はＨｉｎｄ　ＩＩＩ部位）及び、３’―ＲＡＣＥ　ＰＣＲにより得られた断片の配列から設計した、配列番号７記載の終止コドンを含むプライマーｒａｔＥｌｏＲ１（５’−ＧＣＴｔｃｔａｇａＣＴＡＣＴＣＧＧＣＣＴＴＣＧＴＣＧＣＴＴＴＣＴＴ−３’；小文字部分はＸｂａ　Ｉ部位）を用いて以下のようにＰＣＲを行った。なお、高フィデリティーを得るため、ＫＯＤポリメラーゼを用いた。
【００４４】

ＰＣＲプログラム
▲１▼９４℃　２分
▲２▼９４℃　１５秒
▲３▼６０℃　３０秒
▲４▼６８℃　１分　　▲２▼〜▲４▼を３５サイクル
このＰＣＲにより得られた約８５０　ｂｐの遺伝子断片をシークエンス解析した結果、目的の遺伝子であることがわかった。
【００４５】
《ｒＥＬＯ２の開始コドン周辺の配列決定》
ｒＥＬＯ２の全長単離に用いたプライマーｒａｔＥｌｏＦ１はマウス由来の配列であるので、この部分についてはラット遺伝子の配列を決定する必要がある。そこでＡＷ４７５６８１の配列を元に、ｒａｔＥｌｏＦ１部分よりさらに５’上流側（開始コドン相当配列より約１１０　ｂｐ上流）の配列に対応する配列番号１６記載のプライマー　５ＡＷ４７５Ｆ１（５’−ＧＣＣＴＴＣＡＴＴＴＣＣＴＣＣＴＡＣＧＧ−３’）を設計し、以下のようにＰＣＲを行った。
【００４６】

ＰＣＲプログラム
▲１▼９５℃　２分
▲２▼９５℃　１分
▲３▼５５℃　１分
▲４▼７２℃　１分　　▲２▼〜▲４▼を３５サイクル
▲５▼７２℃　１０分
このＰＣＲにより約６００　ｂｐのＤＮＡ断片の増幅が確認できたので、シークエンス解析を行ったところ、目的の遺伝子であることが確認された。開始コドン周辺部分の塩基配列を精査した結果、マウスの配列を元にして作製したｒａｔＥｌｏＦ１プライマーの配列のうち開始コドンＡＴＧのＡから１７番目のＧが、ラットにおいてはＴであることがわかった。したがって、ラットにおいては配列番号２記載のアミノ酸配列におけるアミノ酸番号６のロイシンがマウスの該部位ではトリプトファンである。
【００４７】
［酵母における遺伝子発現］
それぞれｒＥＬＯ１およびｒＥＬＯ２をコードするＰＣＲ産物を、Ｈｉｎｄ　ＩＩＩおよびＸｂａ　Ｉで処理して、ガラクトース誘導性のプロモーターＧＡＬ１の支配下で導入遺伝子を発現する酵母発現ベクターｐＹＥＳ２（Ｉｎｖｉｔｒｏｇｅｎ、　サンディエゴ、　ＵＳＡ）の対応する制限酵素認識部位にライゲーションした。全ての挿入ＤＮＡの配列を、ＤＹＥｎａｍｉｃ　ＥＴ　ターミネーター・サイクル・シークエンシング・キット（Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ　Ｂｉｏｔｅｃｈ、　Ｕｐｐｓａｌａ、　スウェーデン）、及び、ジェネッティックアナライザー（タイプ３１０、　Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社、　フォスターシティー、　ＵＳＡ）による連鎖停止シークエンシングを用いて分析した。
【００４８】
前記プラスミドを、酵母（Ｓ．　ｃｅｒｅｖｉｓｉａｅ）株であるＩＮＶＳｃ１株（α／ａ，　ｈｉｓ３／ｈｉｓ３，　ｌｅｕ２／ｌｅｕ２，　ｔｒｐ１／ｔｒｐ１，　ｕｒａ３／ｕｒａ３；　Ｉｎｖｉｔｒｏｇｅｎ社から購入）、又は、ＯＬＥ１（Δ９不飽和化酵素）欠損変異株Ｌ８―１４Ｃ（ａ，　ｏｌｅ１Δ：：　ＬＥＵ２，　ｕｒａ３，　ｈｉｓ４、ラトガーズ大学のＣ．　Ｅ．マーティンから提供された）［１５］に導入した。
【００４９】
酵母形質転換体を、ウラシルが欠損する培地プレート上で選択した。選択、維持、および生育に不飽和脂肪酸を要求するｏｌｅ１欠損変異体由来の形質転換体の初期培養時には、培地にそれぞれ０．５ｍＭのＣ１６：１ｎ―７およびＣ１８：１ｎ―９を添加した。前記酵母形質転換体を、４％ラフィノース、アミノ酸非含有の０．７％酵母窒素ベース（Ｄｉｆｃｏ　Ｌａｂｏｒａｔｏｒｉｅｓ、　デトロイト、　ＵＳＡ）、１％　ｔｅｒｇｉｔｏｌ型　ＮＰ―４０、２０μｇ／ｍｌ　ヒスチジン、６０μｇ／ｍｌ　ロイシン、及び、４０μｇ／ｍｌ　トリプトファンを含む培地５０ｍｌ中で２８℃、一晩培養した。ここで最後に述べた２成分はｏｌｅ１欠損株には使用しなかった。
【００５０】
そして、〜５ｘ１０^６　細胞／ｍｌの細胞密度に達する直前に培養液に０．１ｍＭ（ｏｌｅ１欠損株には０．５ｍＭ）の終濃度で脂肪酸基質を添加した。導入遺伝子の発現を、２％（ｗ／ｖ）までのガラクトースの添加、及び約３０時間までのさらなる培養によって行った。ｏｌｅ１欠損の遺伝的背景を有する系統においては、前記遺伝子の過剰発現が前記宿主の生長阻害を引き起こすことから、誘導期間を最高１０時間に設定した［１５］。
【００５１】
［脂肪酸分析］
酵母細胞を回収し、脱イオン水で洗浄し、そして、脂質画分を以前に記載のようにクロロホルム／メタノールの混合液（２：１、　ｖ／ｖ）で抽出した［１６］。
総脂質画分を、１０％　メタノリック・ハイドロクロライド（ｍｅｔｈａｎｏｌｉｃ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、　東京化成、　東京、　日本）で処理した。メチル化された脂肪酸をヘキサン中に抽出し、フレーム・イオン化検出器を備えたガス―液体クロマトグラフ（ＧＣ、モデル　ＧＣ―１７Ａ、　島津、　京都、　日本）にアプライした。異なる位置に二重結合を有する不飽和脂肪酸異性体を分離し得るＴＣ―７０キャピラリーカラム（ＧＬサイエンス、　東京、　日本）上で昇温法（１℃／分で１９０〜２００℃）を用いてクロマトグラフィを行った。
【００５２】
脂肪酸メチルエステル標準液のピークを指標にしたソーティング、及び、以前に記載［１７］のようにマススペクトル分析に連結されたＧＣ（ＧＣ―ＭＳ、　ＧＣ　Ｍａｔｅ、　ＪＥＯＬ、　東京、　日本）を使用した電子イオン化マススペクトルの比較分析により各々のピークを同定した。比較される標準脂肪酸が利用できなかった場合には、４，４―ジメチルオキサゾリン　（ＤＭＯＸ、　４，４―ｄｉｍｅｔｈｙｌｏｘａｚｏｌｉｎｅ）誘導体をアシル鎖の二重結合の位置を同定するために使用した［１８］。
【００５３】
脂肪酸メチルエステルを２−アミノ―２―メチル１−プロパノールと簡単に混和して、１８０℃で１８時間加熱した。ジクロロメタンによる抽出、及び、脱イオン水による二度の洗浄後、ＤＭＯＸ誘導体をメチルエステルに対する方法と同様の条件下でＧＣ−ＭＳ（ガス‐液体クロマトグラフィ−マススペクトル分析）により分析した。
【００５４】
Ｓｐｒａｇｕｅ―Ｄａｗｌｅｙ系統の雄ラット（〜１６０ｇ、　週齢）を、チャールズリヴァー日本（横浜、日本）から購入し、個々にケージに収容して約５日間随意に水及び食糧を与えた。
【００５５】
飼育環境に順応した後に３匹のラットを４８時間絶食させ、カロリー計算上、６３．９％の炭水化物、１６．９％の脂肪、及び１９．２％の蛋白質、ビタミン及びミネラルから成るコントロール食餌（ＡＩＮ―９３Ｇ、　オリエンタル酵母）を供給した。２４時間の食餌供給後に、ラットを再度４８時間絶食させた。そして、各々のラットにコントロール食餌中のダイズ油及びカゼインが、コーンスターチ及び脱脂カゼインに置き換えられた無脂肪食を食餌供給した。
【００５６】
その後、ラットを屠殺して、肝臓及び他の器官を迅速に摘出した。コントロール・ラットには前記期間にわたってコントロール食餌を供給し、各器官を上記と同様に摘出した。肝臓の総脂質をＦｏｌｃｈ等の方法［１９］により抽出し、脂肪酸組成を上記の通りに分析した。
【００５７】
［ＲＮＡプローブ及びノーザンブロット分析］
ｒＥＬＯ１（１―３６７ｂｐ）、及び、ｒＥＬＯ２（１―４２９ｂｐ）のｃＤＮＡを含むＤＮＡ断片を、ｐＢｌｕｅｓｃｒｉｐｔ　ＳＫ（＋）（Ｓｔｒａｔａｇｅｎｅ、　ラ・ホーヤ、　ＵＳＡ）ベクターにライゲーションして、ｃＤＮＡのすぐ上流のＨｉｎｄ　ＩＩＩ部位で消化して線状化した。ラン・オフ・アンチセンス転写産物を、フルオレセインＲＮＡ標識混合物（ロシュ・ダイアゴニスティックス、　バーゼル、　スイス）、及び、線状化ＤＮＡ鋳型の存在下でＳＰ６またはＴ７　ＲＮＡポリメラーゼを使用して産生した。ラットのステアロイルＣｏＡ不飽和化酵素１（ＳＣＤ１）に対するＲＮＡプローブは、そのオープンリーディングフレームの１０３―１１７７　ｂｐに対応する領域を含むものである。β―アクチン遺伝子（５１３ｂｐの鎖長）の検出強度をもとにノーザン・ブロットのノーマライゼーション行った。前記プローブを使用の直前に５分間煮沸して変性させた。
【００５８】
ポリアデニル化したＲＮＡを抽出し、製造業者の指示どおりにμＭＡＣＳ　ｍＲＮＡ単離キット（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ、　ドイツ）でラット組織から精製した。前記ｍＲＮＡを、１％のアガロース／ホルムアルデヒド・ゲルで分析し、それからＴｕｒｂｏｂｌｏｔｔｅｒシステム（Ｓｃｈｌｅｉｃｈｅｒ＆Ｓｃｈｕｅｌｌ、　Ｋｅｅｎｅ、　ＵＳＡ）を使用してハイボンド―Ｎ　ナイロン膜（Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ　Ｂｉｏｔｅｃｈ）に転写した。前記ブロット膜を、ＥｘｐｒｅｓｓＨｙｂハイブリダイゼーション緩衝液（Ｃｌｏｎｔｅｃｈ、　パロアルト、　ＵＳＡ）中で１時間７０―７５℃でプレハイブリダイズした。
【００５９】
４ｎｇ／ｍｌの変性フルオレセイン標識ＲＮＡプローブを含む同様の緩衝液中で前記ブロット膜を７０〜７５℃で一晩ハイブリダイズした。ハイブリダイゼーションの後、ブロットをＧｅｎｅ　Ｉｍａｇｅｓ　ＣＤＰ―Ｓｔａｒ検出モジュール（Ａｍｅｒｓｈａｍ　Ｐｈａｒｍａｃｉａ　Ｂｉｏｔｅｃｈ）を使用して免疫化学的にフジＲＸフィルム上で検出した。
【００６０】
＜結果＞
［脂肪酸延長酵素をコードする２つの哺乳動物遺伝子のクローン化］
ｒＥＬＯ１及びｒＥＬＯ２のｃＤＮＡのヌクレオチド配列は、それぞれ２９９及び２６７残基のアミノ酸ポリペチドをコードするＯＲＦを含むものであった。図１は、それらの推定アミノ酸配列と、他の哺乳類の延長酵素及び延長酵素類似蛋白質の配列とを整列させて比較したものである。
【００６１】
図１において、ｒＥＬＯ１（ＤＤＢＪアクセッション番号ＡＢ０７１９８５）及びｒＥＬＯ２（ＡＢ０７１９８６）のｃＤＮＡ配列をもとに翻訳したアミノ酸配列を、ＨＥＬＯ１（ＡＦ２３１９８１）、ｒＥＬＯ２のヒト・ホモログ（ｈＥＬＯ２、　ＡＫ０２７０３１）、及びマウス延長酵素であるＣｉｇ３０（ｍＣｉｇ３０、　Ｕ９７１０７）及びＳｓｃ１（ｍＳｓｃ１、　ＡＦ１７０９０７）の推測上のアミノ酸配列と共に整列させた。ｒＥＬＯ２と同一の残基は白黒を反転表示した。保存されたヒスチジン・ボックスには下線を引いた。アラインメントの上に付点を付けられた列は、ＴＭＰｒｅｄｉｃｔアルゴリズム分析［３３］によって、強い膜貫通ヘリックスを形成する可能性が高いことが推測されるｒＥＬＯ２配列の領域を示している。
【００６２】
ｒＥＬＯ１配列は、ヒト延長酵素　ＨＥＬＯ１（９３．０％同一）の配列に対して最も高い相同性を有しており、続いてマウス延長酵素類似蛋白質　Ｓｓｃ２（５７．４％；　配列示さず）、マウス延長酵素　Ｓｓｃ１（３４．３％）、Ｃｉｇ３０（２５．８％）の順番で相同性を有している。
【００６３】
また、ｒＥＬＯ２配列は、本発明においてｈＥＬＯ２と命名されたヒトの未知の蛋白質（ＡＫ０２７０３１）（９５．８％）をコードする配列、及び、マウスＣｉｇ３０（４２．９％）と類似していた。
【００６４】
ｒＥＬＯ１及びｒＥＬＯ２間の相同性スコアは２５．０％であった。アラインメントによって、全ての配列が脂肪酸不飽和化酵素［２０］及び延長酵素［１］（図１）を含む鉄含有酵素間で保存されている特徴的なＨＸＸＨＨモチーフ（配列番号１７に記載；Ｘは任意のアミノ酸を意味する）を共有していることが示された。これらの配列はＧＳＡモチーフ（ＮＡＤＰＨ−結合部位であると提唱されている）を欠いている。また、ＧＳＡモチーフは酵母ＥＬＯ１の配列中に認められたが、酵母ＥＬＯ２及び酵母ＥＬＯ３には認められなかった［１０］。
【００６５】
ｒＥＬＯ１と同様にｒＥＬＯ２ポリペチドの疎水性プロットのパターンは、以前に報告され５つの膜貫通領域の存在が予測された［９−１１］延長酵素のパターンと類似性を示した（図１）。したがって、ｒＥＬＯ１はＨＥＬＯ１のラット・ホモログであり、そしてｒＥＬＯ２及びそのヒト・ホモログ　ｈＥＬＯ２は、脂肪酸延長酵素ファミリーの新規メンバーであることが解明された。
【００６６】
［酵母におけるｒＥＬＯ１及びｒＥＬＯ２遺伝子の機能的発現］
ラット延長酵素と推測される遺伝子の機能を調査及び比較するために、前記遺伝子を酵母（Ｓ．　ｃｅｒｅｖｉｓｉａｅ　ＩＮＶＳｃ１、　野生型）に導入して発現誘導した。脂肪酸を添加しない条件下において、ｒＥＬＯ１遺伝子の発現は顕著量のＣ１８：１ｎ―７を新規に生成した（表１）。この結果は、ＨＥＬＯ１（ｒＥＬＯ１のヒト・ホモログ）を酵母で発現した［１４］際に観察される現象と同様であり、内因性のＣ１６：１ｎ―７が２−炭素ユニット延長したことを示唆している。
【００６７】
【表１】

【００６８】
同様に、少量検出されるエイコセン酸（Ｃ２０：１ｎ―７及びＣ２０：１ｎ―９）は、それぞれ生成されたＣ１８：１ｎ―７及び内因性のＣ１８：１ｎ―９に由来する延長産物であると考えられた。
【００６９】
一方、野生型酵母でのｒＥＬＯ２遺伝子の発現は、ｒＥＬＯ１発現（表１）の場合と対照的に極めて微量のＣ１８：１ｎ―７（４．０％）を生成する一方で，Ｃ１８：　１ｎ―９含量（７１．０％）を著しく増加させた。これらＣ１８：１異性体上の二重結合の位置の相違により、我々はそれら生成物の由来を区別し得る、すなわち、上記のようにＣ１８：１ｎ―７はｒＥＬＯ１の作用によってＣ１６：１ｎ―７から転換されたものであり、一方、Ｃ１８：１ｎ―９は内在性の酵素（ＯＬＥ１ｐ）によるΔ９不飽和化を介してＣ１８：０から転換されたものである。
【００７０】
Ｃ１６：１ｎ―７が顕著に増加しなかったという事実は、内因性のＯＬＥ１ｐ活性がｒＥＬＯ２誘導によって増加した可能性を否定するものである。従って、我々の結果はｒＥＬＯ２発現によるＣ１８：１ｎ―９の増加がＣ１６：０の鎖延長反応の活性化によるものであることを示すものである。
【００７１】
さらに、ｒＥＬＯ２の過剰発現により、Ｃ１８：１ｎ―７と同様の微量レベルではあるが、Ｃ２０：０、及びそのΔ９―不飽和化産物であるＣ２０：１ｎ―１１に対応するピークを示した。しかし、Ｃ２０：１ｎ―９またはＣ２０：１ｎ―７は検出されなかった（表１）。従って、全ての検出可能なモノ不飽和脂肪酸は、Δ９位に二重結合を有し、対応する飽和種に由来するものである。
【００７２】
ｒＥＬＯ１及びｒＥＬＯ２の作用をより明確に示すために、一連の発現実験を酵母変異体（Ｌ８―１４Ｃ）を使用して行った。ＯＬＥ１（Δ９不飽和化酵素）遺伝子が破壊されているためにこの変異体においては飽和脂肪酸は不飽和化されない。ｏｌｅ１Δ遺伝子型の変異体は通常の成育に不飽和脂肪酸類を必要とするので、遺伝子誘導する培養培地中に０．５ｍＭのＣ１６：１ｎ―７を添加した。コントロール（ｐＹＥＳ２、　表２）でのＣ１８：１ｎ―７の微量の産生は、Ｃ１６：１ｎ―７に作用する現在未確認の内在性の延長酵素活性によるものかもしれない。
【００７３】
【表２】

【００７４】
Ｃ１８：１ｎ―９が検出されなかったことは、ＯＬＥ１ｐの不活性化を証明するものであった。このような条件下において、ｒＥＬＯ１の発現は、コントロール（２．９％）と比較してＣ１６：１ｎ―７含量を若干減少させたものの，Ｃ１８：１ｎ―７（２０．３％）を著しく増加させた（表２）。他方、ｒＥＬＯ２発現は、Ｃ１８：０の増加及びＣ１６：０を減少させ、そのため飽和脂肪酸の組成においてＣ１８：０／Ｃ１６：０の割合がコントロール（１．０９）及びｒＥＬＯ１場合（１．１５）と比較して非常に高い２．６７という値にまで顕著に増加させた。この結果は、ｒＥＬＯ２の飽和脂肪酸指向性の延長活性を追認するものであった。
【００７５】
Ｃ２０：０は検出可能な水準以下であった（データ示さず）が、この結果はＣ１８：０基質が少量であったため、及び、ｒＥＬＯ２活性がｏｌｅ１Δの遺伝的背景において顕著でなかったためである可能性がある。延長酵素に類似の構造（図１）を有していることを考え合わせても、以上の結果はｒＥＬＯ２が主にＣ１６：０、及び、より低レベルでＣ１８：０に作用する脂肪酸延長酵素であるという結論を支持するものである。
【００７６】
Ｃ１８：１ｎ―７含量がｒＥＬＯ２を発現した酵母（表１及び２）において多少増加した点にも注目した。これはｒＥＬＯ２の広い基質特異性を示唆する結果である。ｒＥＬＯ１もＣ１６：　１ｎ―７からＣ１８：　１ｎ―７への延長反応（表１及び２）を触媒したので、２つの延長酵素の基質範囲はオーバーラップすると考えられた。ｒＥＬＯ１、またはｒＥＬＯ２遺伝子を保持する野性型酵母形質転換株を使用して、その可能性を検証した。
【００７７】
表３に要約された結果に見られるように、ｒＥＬＯ１発現はＣ１８：４ｎ―３、次いでＣ１８：３ｎ―６に対して高い転換割合を示した。これらのケースの各々において、それぞれＣ２２：　４ｎ―３及びＣ２２：　３ｎ―６の低レベルの生成が観察された。この結果は生成された脂肪酸（Ｃ２０：４ｎ―３及びＣ２０：３ｎ―６）が更に延長されたことを示すものである。ｒＥＬＯ１の発現によって各々の２炭素延長された産物は、アラキドン酸（Ｃ２０：４ｎ―６）及びＣ２０：５ｎ―３に加えて、リノール酸（Ｃ１８：２ｎ―６）及びα―リノレン酸（Ｃ１８：３ｎ―３）の存在下でさえ生成した。
【００７８】
【表３】

【００７９】
ジホモ―γ―リノレン酸（Ｃ２０：３ｎ―６）、及び、ミード酸（Ｃ２０：３ｎ―９）に対する転換割合は３％未満であった。この他、ｒＥＬＯ２発現の場合、Ｃ１８　ＰＵＦＡがそれぞれ対応するＣ２０　ＰＵＦＡに延長されたが、その延長反応は低レベル（３―７％）であり、Ｃ２０　ＰＵＦＡの更なる転換は観察し得なかった。同条件下でｐＹＥＳ２を保持するコントロール酵母を発現誘導しても、いかなる延長産物も生成しなかった（データ示さず）。
【００８０】
これらの結果は、長鎖ＰＵＦＡに関してＣ１８及びＣ２０　ＰＵＦＡの延長がｒＥＬＯ１により触媒され、そして、軽微なレベルのＣ１８不飽和物の延長がｒＥＬＯ２によっても行われることを示唆するものである。ｒＥＬＯ１及びｒＥＬＯ２とも、Ｃ２２　ＰＵＦＡに対して延長活性を呈さなかった。
【００８１】
［延長酵素の遺伝子発現の食餌制御］
ラット肝臓ミクロソームにおいて、Ｃ１６：０に対する総延長活性（Δ９不飽和化活性と同様に）は絶食によって低下し、通常食または無脂肪食の再給餌により迅速に高水準にまで上昇すると報告されている［５、２１］。
【００８２】
アクチノマイシンＤ（ＲＮＡ転写阻害剤）が鎖延長反応における再給餌効果を抑制したので［２１］、延長活性の増大は遺伝子発現の誘導によって引き起こされるのかもしれない。この可能性を検証するために、肝臓遺伝子のノーザン分析をｒＥＬＯ１、ｒＥＬＯ２及びＳＣＤ１遺伝子に相補的なＲＮＡプローブを使用して行った。図２において、（Ａ）はノーザン分析を、ｒＥＬＯ２、ｒＥＬＯ１、ＳＣＤ１またはβ−アクチンに相補的なＲＮＡプローブを使用して、コントロール（ＣＴ）及びＦＦＤラットの各々３匹から抽出した肝臓のｍＲＮＡ（０．２μｇ／レーン）に対して行った結果である。バンドの推定サイズをキロベース（ｋｂ）として示した。（Ｂ）は（Ａ）のバンドの検出シグナル強度をデンシトメトリで測定した数値をグラフで示したものである。各値は、β−アクチンの測定値に対する各々の測定値の水準の平均値に標準偏差を付して示した。「＊」は対応するコントロールより顕著に高い（ｐ＜０．０１）ものに付した。この実験は３回反復し、その何れにおいても同様の結果が示された。図２に示されるように、ｒＥＬＯ２及びＳＣＤ１転写産物の量は、ラットの絶食及び再給餌スケジュール（ＦＦＤ）の２回の繰り返しによって４〜５倍にまで増加した。対照的にＦＦＤ処理は、ｒＥＬＯ１の発現にほとんど影響しなかった。この結果は２つの延長酵素の発現制御機構における相違を示唆している。
【００８３】
以前のデータと同様に、ラットのＦＦＤ処理は肝臓の脂肪酸組成に対して顕著な影響を及ぼした。即ち、Ｃ１６：　１ｎ―７及びＣ１８：　１ｎ―９の含量は、コントロール・ラットと比較して、それぞれ、５倍及び２倍増加した（表４）。この結果は報告されているようにＳＣＤ１の活性化を示すものである［２２―２４］。
【００８４】
【表４】

【００８５】
この際、　Ｃ１８：０含量は若干減少（５６％にまで）した。しかし、デノボ経路のＣ１８脂肪酸（Ｃ１８：　０及びＣ１８：　１ｎ―９）の量は１．３倍まで上昇した。これはＦＦＤ処理によるｒＥＬＯ２誘導の結果であるかもしれない。一方、ｎ―６及びｎ―３系の主要なＰＵＦＡの含量は若干減少するか又は不変であった。ＰＵＦＡ組成のいかなる変動も、ｒＥＬＯ１の活性化または抑制を示唆するものではなかった。これらのデータは、ｒＥＬＯ１及びｒＥＬＯ２活性の発現がＦＦＤ処理によって異なる様式で制御されることを更に示唆するものである。
【００８６】
ＦＦＤラットのノーザン分析によって遺伝子発現の組織分布を調べた。図３においては、図の上側に示したＦＦＤラットの各器官のｍＲＮＡサンプル（０．２μｇ／レーン）を使用したノーザン分析を、ｒＥＬＯ１、ｒＥＬＯにまたはβ−アクチンに対するＲＮＡプローブを使用して行った。この実験は３回反復し、その何れにおいても同様の結果を得た。結果として，ｒＥＬＯ１が多様な組織において発現していることがわかり、肺臓及び脳（図３）においては最も高いレベルで発現していることがわかった。この結果は精巣での結果を除いては以前の報告［１４］と同様であった。対照的に、ｒＥＬＯ２の発現は主に肝臓において、及び、わずかに脳において観察されたが、他の組織（図３）、又は、コントロール・ラット（データ示さず）の全ての検査した組織においては観察されなかった。ｒＥＬＯ２の肝細胞における発現は、ＦＦＤ処理で抑制された長鎖脂肪酸の迅速な合成に寄与し得ると考えられた。
【００８７】
ワックス［９］、及び、中鎖／長鎖脂肪酸［１０―１４］の生合成に関与する脂肪酸延長酵素の分子同定における最近の進歩は、それら酵素の構造上の特性を明らかにした。それらの特性には、ＨＸＸＨＨ（配列番号１７）として表現される特徴的なモチーフを有することなどが含まれ、前記モチーフは脂肪酸不飽和化酵素及び炭化水素ヒドロキシラーゼのようなジイオン−オキソ（ｄｉｉｏｎ―ｏｘｏ）酵素［２０］に認められているものである。
【００８８】
保存されたモチーフのヒスチジン残基は、酸素がＯ_２−依存的レドックス反応の電子移動に寄与するＦｅ―Ｏ―Ｆｅクラスタを形成するＦｅ―キレーティングリガンドとして作用すると信じられている［２５］。
【００８９】
延長酵素蛋白質の別の特性としては、膜貫通領域と予測される５つの疎水性のストレッチが存在することが挙げられる。この特性は、主にミクロソーム膜画分において脂肪酸延長活性が局在するとの知見と一致するものである。しかし、これらの構造上の特色は、それぞれ、Ｃ１６：０、及び、より長鎖の飽和脂肪酸の鎖延長における縮合反応を触媒するＫＡＳ　ＩＩ［２６］、及び、ＦＡＥ１［２７］を含む植物酵素では認められていない。この事実はそれら植物酵素の反応スキームにおける異なる機構の存在を示唆するものである。この機構の違いは，触媒し得る脂肪酸基質の誘導体群の構造上の相違に関係する可能性がある。実際に、植物酵素は基質としてアシルキャリア蛋白質またはグリセリドに結合する脂肪酸を利用するが、動物（および、おそらく酵母を含む真菌類）の延長酵素は、コエンザイムＡ―活性化基質を要求する。Ｃ１８：　０合成に関与する縮合酵素に関して、本発明で同定された哺乳類延長酵素　ｈＥＬＯ２、及び、ｒＥＬＯ２は後者の最初の例であって、植物酵素とは構造的類似性を有さないものである。
【００９０】
本発明において、ｒＥＬＯ２のＣ１６：　０による延長活性を示すために酵母の異種宿主発現系を利用した。野性型の酵母を宿主として使用した際に、ｒＥＬＯ２の過剰発現はＣ１８：　１ｎ―９含量の著しい増加を生じた（表１）。実験系からＯＬＥ１ｐの関与を排除するため、次にｏｌｅ１欠損酵母変異体Ｌ８―１４ＣにおいてｒＥＬＯ２の発現系を構築し、遺伝子発現誘導によりＣ１８：　０の増加を観察した（表２）。しかし、変異体のＣ１６：　０（２．６７）に対するＣ１８：　０の割合は、野性型宿主で観察された割合ほど高いものではなかった（７．３２、　表１）。この結果は、ｏｌｅ１Δ宿主に対するガラクトースの添加後の培養時間を、野生型宿主の場合より短く設定したためであると思われた。遺伝子の過剰発現に対してｏｌｅ１Δ宿主が不安定であることから、培養時間の制限を回避することができない［１５］。これに加えて、多量に加わられたｒＥＬＯ２のマイナーな基質であるＣ１６：　１ｎ―７がＣ１６：０の競争者となったのかもしれない。
【００９１】
ＰＵＦＡに対するｒＥＬＯ１及びｒＥＬＯ２の基質特異性について調べた結果、ｒＥＬＯ１がＣ１８：　４ｎ―３及びＣ１８：　３ｎ―６を延長する顕著に高い活性を有することがわかった（表３）。また以前の報告と同様に、Ｃ２０：　５ｎ―３、Ｃ１８：　３ｎ―３、Ｃ２０：　４ｎ―６及びＣ１８：　２ｎ―６を含む他のＰＵＦＡは、中程度または低程度の転換率で延長された［１４］。
【００９２】
同じ鎖長の脂肪酸と比較して、不飽和度が高い基質及びｎ―３位置に二重結合を有する基質は、ｒＥＬＯ１によって優先的に延長される傾向にあった。主要なｎ―３　ＰＵＦＡの鎖長（Ｃ２０：５ｎ―３及びＣ２２：６ｎ―３）が主要なｎ―６　ＰＵＦＡ（Ｃ１８：２ｎ―６及びＣ２０：４ｎ―６）より長いという脂肪酸分布を示す哺乳動物細胞において、このようなｎ―３　ＰＵＦＡに対する高い選択性は、Ｃ１８、Ｃ２０及びＣ２２　ＰＵＦＡの含量割合を部分的に反映しているのかもしれない。一方、ｒＥＬＯ２の基質特異性はＣ１８　ＰＵＦＡの転換を低レベルながら触媒するが，ｒＥＬＯ１の基質特異性とは対照的なものであった。
【００９３】
これらの結果は、延長酵素の基質選択性はさほど厳密ではなく、基質のアシル基の鎖長、及び、不飽和度の双方により特異性が異なることを強調するものである。また、ｒＥＬＯ１が飽和脂肪酸に作用しなかった点に留意するべきであり、この事実はＣ１６：　０の延長反応におけるｒＥＬＯ２の重要性を示唆している。さらに、延長酵素のいずれもがＣ２２　ＰＵＦＡに対して検出可能なレベルの延長活性を示さなかったという事実は、未同定の延長酵素の存在を示唆するものである。
【００９４】
Ｎａｋａｇａｗａ等［２２］は、肝臓ミクロソームのＣ１６：　０―コエンザイムＡの延長（ｒＥＬＯ２によると思われる）及びΔ９不飽和化（ＳＣＤ１による）の双方が、どのようにラットの絶食及び再給餌によって活性化されるかを記載している。ＳＣＤ１の活性化は、２−３回の高炭水化物／低脂肪食の食餌スケジュールの反復によって更に増強した［２８］。
【００９５】
本発明において、我々は同様の条件下において肝細胞のＳＣＤ１及びｒＥＬＯ２転写産物量が、それぞれのコントロールより４―５倍増大していることを観察した（図２）。コレステロールを含む脂質の場合と同様に、これらの酵素活性が血糖及びインシュリン量の変化を介した栄養状態及びホルモン状態により調整されることは以前から知られている［１、２３、２９］。これらの栄養的パラメーターの変化も、Ｃ１６：０を供給する脂肪酸シンターゼの遺伝子発現の変調を生じる［３０］。
【００９６】
最近の研究は、食餌脂質の長期間欠乏後の再給餌処理におけるステロール制御因子結合蛋白質１の活性化を介したＳＣＤ１転写の制御機構の存在を示唆している［３１、３２］。ｒＥＬＯ２発現が、ＳＣＤ１調節に関与する因子に共通の細胞内情報伝達によって少なくとも部分的に制御されることは充分考えられる。これらの脂質合成系酵素とは対照的に、ラットのＦＦＤ処理はｒＥＬＯ１の発現を肝細胞で顕著に誘導しなかった（図２）。この結果は、ミクロソームにおけるＣ１８：　３ｎ―６に対する延長活性が、上記のような制限された栄養状態により影響されなかったという以前のデータと一致するものである［５］。
【００９７】
これらのデータは、細胞の飽和及び不飽和脂肪酸のホメオスタシスにおけるデノボ脂肪酸及びＰＵＦＡの生合成系の制御における新規の機構の存在を示唆するものである。
【００９８】
【発明の効果】
以上詳述したように、本発明によれば、飽和脂肪酸の鎖長を酵素活性を利用して延長できる。より具体的には、本発明の酵素蛋白質による飽和脂肪酸特異的な鎖長延長反応を介して、Ｃ１８：０飽和脂肪酸をＣ２０：０飽和脂肪酸にすることができる等、顕著な効果を得ることができる。
【００９９】
【参照文献】
１）　Ｃｉｎｔｉ，　Ｄ．　Ｌ．，　Ｃｏｏｋ，　Ｌ．，　Ｎａｇｉ，　Ｍ．　Ｎ．，　ａｎｄ　Ｓｕｎｅｊａ，　Ｓ．　Ｋ．，　Ｔｈｅ　ｆａｔｔｙ　ａｃｉｄ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｓｙｓｔｅｍ　ｏｆ　ｍａｍｍａｌｉａｎ　ｅｎｄｏｐｌａｓｍｉｃ　ｒｅｔｉｃｕｌｕｍ．　Ｐｒｏｇ．　Ｌｉｐｉｄ　Ｒｅｓ．，　３１，　１−５１　（１９９２）．
２）　Ｃｏｏｋ，　Ｈ．　Ｗ．，　Ｆａｔｔｙ　ａｃｉｄ　ｄｅｓａｔｕｒａｔｉｏｎ　ａｎｄ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｉｎ　ｅｕｋａｒｙｏｔｅｓ．　Ｉｎ　”Ｂｉｏｃｈｅｍｉｓｔｒｙ　ｏｆ　Ｌｉｐｉｄｓ，　Ｌｉｐｏｐｒｏｔｅｉｎｓ　ａｎｄ　Ｍｅｍｂｒａｎｅｓ”，　ｅｄｓ．　Ｖａｎｃｅ，　Ｄ．　Ｅ．　ａｎｄ　Ｖａｎｃｅ，　Ｊ．　Ｅ．，　Ｅｌｓｅｖｉｅｒ　Ｓｃｉｅｎｃｅ，　Ａｍｓｔｅｒｄａｍ，　ｐｐ．　１２９−１５２　（１９９６）．
３）　Ｎｕｇｔｅｒｅｎ，　Ｄ．　Ｈ．，　Ｔｈｅ　ｅｎｚｙｍｉｃ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｆａｔｔｙ　ａｃｉｄｓ　ｂｙ　ｒａｔ−ｌｉｖｅｒ　ｍｉｃｒｏｓｏｍｅｓ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ，　１０６，　２８０−２９０　（１９６５）．
４）　Ｂｅｒｎｅｒｔ，　Ｊ．　Ｔ．，　Ｊｒ．　ａｎｄ　Ｓｐｒｅｃｈｅｒ，　Ｈ．，　Ａｎ　ａｎａｌｙｓｉｓ　ｏｆ　ｐａｒｔｉａｌ　ｒｅａｃｔｉｏｎｓ　ｉｎ　ｔｈｅ　ｏｖｅｒａｌｌ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｓａｔｕｒａｔｅｄ　ａｎｄ　ｕｎｓａｔｕｒａｔｅｄ　ｆａｔｔｙ　ａｃｉｄｓ　ｂｙ　ｒａｔ　ｌｉｖｅｒ　ｍｉｃｒｏｓｏｍｅｓ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２５２，　６７３６−６７４４　（１９７７）．
５）　Ｓｐｒｅｃｈｅｒ，　Ｈ．，　Ｔｈｅ　ｉｎｆｌｕｅｎｃｅ　ｏｆ　ｄｉｅｔａｒｙ　ａｌｔｅｒａｔｉｏｎｓ，　ｆａｓｔｉｎｇ　ａｎｄ　ｃｏｍｐｅｔｉｔｉｖｅ　ｉｎｔｅｒａｃｔｉｏｎｓ　ｏｎ　ｔｈｅ　ｍｉｃｒｏｓｏｍａｌ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｆａｔｔｙ　ａｃｉｄｓ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ，　３６０，　１１３−１２３　（１９７４）．
６）　Ｐｒａｓａｄ，　Ｍ．　Ｒ．　ａｎｄ　Ｃｉｎｔｉ，　Ｄ．　Ｌ．，　Ｅｆｆｅｃｔ　ｏｆ　ｔｈｅ　ｐｅｒｏｘｉｓｏｍａｌ　ｐｒｏｌｉｆｅｒａｔｏｒ　ｄｉ（２−ｅｔｈｙｌｈｅｘｙｌ）ｐｈｔｈａｌａｔｅ　ｏｎ　ｃｏｍｐｏｎｅｎｔ　ｒｅａｃｔｉｏｎｓ　ｏｆ　ｔｈｅ　ｒａｔ　ｈｅｐａｔｉｃｍｉｃｒｏｓｏｍａｌ　ｆａｔｔｙ　ａｃｉｄ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｓｙｓｔｅｍ　ａｎｄ　ｏｎ　ｏｔｈｅｒ　ｈｅｐａｔｉｃ　ｌｉｐｏｇｅｎｉｃ　ｅｎｚｙｍｅｓ．　Ａｒｃｈ．　Ｂｉｏｃｈｅｍ．　Ｂｉｏｐｈｙｓ．，　２４８，　４７９−４８８　（１９８６）．
７）　Ｐｒａｓａｄ，　Ｍ．　Ｒ．，　Ｎａｇｉ，　Ｍ．　Ｎ．，　Ｇｈｅｓｑｕｉｅｒ，　Ｄ．，　Ｃｏｏｋ，　Ｌ．，　ａｎｄ　Ｃｉｎｔｉ，　Ｄ．Ｌ．，　Ｅｖｉｄｅｎｃｅ　ｆｏｒ　ｍｕｌｔｉｐｌｅ　ｃｏｎｄｅｎｓｉｎｇ　ｅｎｚｙｍｅｓ　ｉｎ　ｒａｔ　ｈｅｐａｔｉｃ　ｍｉｃｒｏｓｏｍｅｓ　ｃａｔａｌｙｚｉｎｇ　ｔｈｅ　ｃｏｎｄｅｎｓａｔｉｏｎ　ｏｆ　ｓａｔｕｒａｔｅｄ，　ｍｏｎｏｕｎｓａｔｕｒａｔｅｄ，　ａｎｄ　ｐｏｌｙｕｎｓａｔｕｒａｔｅｄ　ａｃｙｌ　ｃｏｅｎｚｙｍｅ　Ａ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２６１，　８２１３−８２１７　（１９８６）．
８）　Ｌｕｔｈｒｉａ，　Ｄ．　Ｌ．　ａｎｄ　Ｓｐｒｅｃｈｅｒ，　Ｈ．，　Ｓｔｕｄｉｅｓ　ｔｏ　ｄｅｔｅｒｍｉｎｅ　ｉｆ　ｒａｔ　ｌｉｖｅｒｃｏｎｔａｉｎｓ　ｍｕｌｔｉｐｌｅ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｎｇ　ｅｎｚｙｍｅｓ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ，　１３４６，　２２１−２３０　（１９９７）．
９）　Ｔｖｒｄｉｋ，　Ｐ．，　Ｗｅｓｔｅｒｂｅｒｇ，　Ｒ．，　Ｓｉｌｖｅ，　Ｓ．，　Ａｓａｄｉ，　Ａ．，　Ｊａｋｏｂｓｓｏｎ，　Ａ．，　Ｃａｎｎｏｎ，　Ｂ．，　Ｌｏｉｓｏｎ，　Ｇ．，　ａｎｄ　Ｊａｃｏｂｓｓｏｎ，　Ａ．，　Ｒｏｌｅ　ｏｆ　ａ　ｎｅｗ　ｍａｍｍａｌｉａｎ　ｇｅｎｅ　ｆａｍｉｌｙ　ｉｎ　ｔｈｅ　ｂｉｏｓｙｎｔｈｅｓｉｓ　ｏｆ　ｖｅｒｙ　ｌｏｎｇ　ｃｈａｉｎ　ｆａｔｔｙ　ａｃｉｄｓ　ａｎｄ　ｓｐｈｉｎｇｏｌｉｐｉｄｓ．　Ｊ．　Ｃｅｌｌ　Ｂｉｏｌ．，　１４９，　７０７−７１８　（２０００）．
１０）　Ｏｈ，　Ｃ．　Ｓ．，　Ｔｏｋｅ，　Ｄ．　Ａ．，　Ｍａｎｄａｌａ，　Ｓ．，　ａｎｄ　Ｍａｒｔｉｎ，　Ｃ．　Ｅ．，　ＥＬＯ２　ａｎｄ　ＥＬＯ３，　ｈｏｍｏｌｏｇｕｅｓ　ｏｆ　ｔｈｅ　Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ　ＥＬＯ１　ｇｅｎｅ，　ｆｕｎｃｔｉｏｎ　ｉｎ　ｆａｔｔｙ　ａｃｉｄ　ｅｌｏｎｇａｔｉｏｎ　ａｎｄ　ａｒｅ　ｒｅｑｕｉｒｅｄ　ｆｏｒ　ｓｐｈｉｎｇｏｌｉｐｉｄ　ｆｏｒｍａｔｉｏｎ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２７２，　１７３７６−１７３８４　（１９９７）．
１１）　Ｔｏｋｅ，　Ｄ．　Ａ．　ａｎｄ　Ｍａｒｔｉｎ，　Ｃ．　Ｅ．，　Ｉｓｏｌａｔｉｏｎ　ａｎｄ　ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ　ｏｆ　ａ　ｇｅｎｅ　ａｆｆｅｃｔｉｎｇ　ｆａｔｔｙ　ａｃｉｄ　ｅｌｏｎｇａｔｉｏｎ　ｉｎ　Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２７１，　１８４１３−１８４２２　（１９９６）．
１２）　Ｂｅａｕｄｏｉｎ，　Ｆ．，　Ｍｉｃｈａｅｌｓｏｎ，　Ｌ．　Ｖ．，　Ｈｅｙ，　Ｓ．　Ｊ．，　Ｌｅｗｉｓ，　Ｍ．　Ｊ．，　Ｓｈｅｗｒｙ，　Ｐ．　Ｒ．，　Ｓａｙａｎｏｖａ，　Ｏ．，　ａｎｄ　Ｎａｐｉｅｒ，　Ｊ．　Ａ．，　Ｈｅｔｅｒｏｌｏｇｏｕｓ　ｒｅｃｏｎｓｔｉｔｕｔｉｏｎ　ｉｎｙｅａｓｔ　ｏｆ　ｔｈｅ　ｐｏｌｙｕｎｓａｔｕｒａｔｅｄ　ｆａｔｔｙ　ａｃｉｄ　ｂｉｏｓｙｎｔｈｅｔｉｃ　ｐａｔｈｗａｙ．　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　Ｕ　Ｓ　Ａ，　９７，　６４２１−６４２６　（２０００）．
１３）　Ｐａｒｋｅｒ−Ｂａｒｎｅｓ，　Ｊ．　Ｍ．，　Ｄａｓ，　Ｔ．，　Ｂｏｂｉｋ，　Ｅ．，　Ｌｅｏｎａｒｄ，　Ａ．　Ｅ．，　Ｔｈｕｒｍｏｎｄ，　Ｊ．　Ｍ．，　Ｃｈａｕｎｇ，　Ｌ．　Ｔ．，　Ｈｕａｎｇ，　Ｙ．　Ｓ．，　ａｎｄ　Ｍｕｋｅｒｊｉ，　Ｐ．，　Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ　ａｎｄ　ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ　ｏｆ　ａｎ　ｅｎｚｙｍｅ　ｉｎｖｏｌｖｅｄ　ｉｎ　ｔｈｅ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｎ−６　ａｎｄ　ｎ−３　ｐｏｌｙｕｎｓａｔｕｒａｔｅｄ　ｆａｔｔｙ　ａｃｉｄｓ．　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　Ｕ　Ｓ　Ａ，　９７，　８２８４−８２８９　（２０００）．
１４）　Ｌｅｏｎａｒｄ，　Ａ．　Ｅ．，　Ｂｏｂｉｋ，　Ｅ．　Ｇ．，　Ｄｏｒａｄｏ，　Ｊ．，　Ｋｒｏｅｇｅｒ，　Ｐ．　Ｅ．，　Ｃｈｕａｎｇ，Ｌ．　Ｔ．，　Ｔｈｕｒｍｏｎｄ，　Ｊ．　Ｍ．，　Ｐａｒｋｅｒ−Ｂａｒｎｅｓ，　Ｊ．　Ｍ．，　Ｄａｓ，　Ｔ．，　Ｈｕａｎｇ，　Ｙ．　Ｓ．，　ａｎｄ　Ｍｕｋｅｒｊｉ，　Ｐ．，　Ｃｌｏｎｉｎｇ　ｏｆ　ａ　ｈｕｍａｎ　ｃＤＮＡ　ｅｎｃｏｄｉｎｇ　ａ　ｎｏｖｅｌ　ｅｎｚｙｍｅ　ｉｎｖｏｌｖｅｄ　ｉｎ　ｔｈｅ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｌｏｎｇ−ｃｈａｉｎ　ｐｏｌｙｕｎｓａｔｕｒａｔｅｄ　ｆａｔｔｙ　ａｃｉｄｓ．　Ｂｉｏｃｈｅｍ．　Ｊ．，　３５０，　７６５−７７０　（２０００）．
１５）　Ｓｔｕｋｅｙ，　Ｊ．　Ｅ．，　ＭｃＤｏｎｏｕｇｈ，　Ｖ．　Ｍ．，　ａｎｄ　Ｍａｒｔｉｎ，　Ｃ．　Ｅ．，　Ｉｓｏｌａｔｉｏｎ　ａｎｄｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ　ｏｆ　ＯＬＥ１，　ａ　ｇｅｎｅ　ａｆｆｅｃｔｉｎｇ　ｆａｔｔｙ　ａｃｉｄ　ｄｅｓａｔｕｒａｔｉｏｎ　ｆｒｏｍＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２６４，　１６５３７−１６５４４　（１９８９）．
１６）　Ａｋｉ，　Ｔ．，　Ｎａｇａｈａｔａ，　Ｙ．，　Ｉｓｈｉｈａｒａ，　Ｋ．，　Ｔａｎａｋａ，　Ｙ．，　Ｍｏｒｉｎａｇａ，　Ｔ．，　Ｈｉｇａｓｈｉｙａｍａ，　Ｋ．，　Ａｋｉｍｏｔｏ，　Ｋ．，　Ｆｕｊｉｋａｗａ，　Ｓ．，　Ｋａｗａｍｏｔｏ，　Ｓ．，　Ｓｈｉｇｅｔａ，　Ｓ．，　Ｏｎｏ，　Ｋ．，　ａｎｄ　Ｓｕｚｕｋｉ，　Ｏ．，　Ｐｒｏｄｕｃｔｉｏｎ　ｏｆ　ａｒａｃｈｉｄｏｎｉｃ　ａｃｉｄ　ｂｙ　ｆｉｌａｍｅｎｔｏｕｓ　ｆｕｎｇｕｓ，　Ｍｏｒｔｉｅｒｅｌｌａ　ａｌｌｉａｃｅａ　ｓｔｒａｉｎ　ＹＮ−１５．　Ｊ．　Ａｍ．　Ｏｉｌ．　Ｃｈｅｍ．　Ｓｏｃ．，　７８，　５９９−６０４　（２００１）．
１７）　Ａｋｉ，　Ｔ．，　Ｓｈｉｍａｄａ，　Ｙ．，　Ｉｎａｇａｋｉ，　Ｋ．，　Ｈｉｇａｓｈｉｍｏｔｏ，　Ｈ．，　Ｋａｗａｍｏｔｏ，　Ｓ．，Ｓｈｉｇｅｔａ，　Ｓ．，　Ｏｎｏ，　Ｋ．，　ａｎｄ　Ｓｕｚｕｋｉ，　Ｏ．，　Ｍｏｌｅｃｕｌａｒ　ｃｌｏｎｉｎｇ　ａｎｄ　ｆｕｎｃｔｉｏｎａｌ　ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ　ｏｆ　ｒａｔ　Δ−６　ｆａｔｔｙ　ａｃｉｄ　ｄｅｓａｔｕｒａｓｅ．　Ｂｉｏｃｈｅｍ．　Ｂｉｏｐｈｙｓ．　Ｒｅｓ．　Ｃｏｍｍｕｎ．，　２５５，　５７５−５７９　（１９９９）．
１８）　Ｌｕｔｈｒｉａ，　Ｄ．　Ｌ．　ａｎｄ　Ｓｐｒｅｃｈｅｒ，　Ｈ．，　２−Ａｌｋｅｎｙｌ−４，４−ｄｉｍｅｔｈｙｌｏｘａｚｏｌｉｎｅｓａｓ　ｄｅｒｉｖａｔｉｖｅｓ　ｆｏｒ　ｔｈｅ　ｓｔｒｕｃｔｕｒａｌ　ｅｌｕｃｉｄａｔｉｏｎ　ｏｆ　ｉｓｏｍｅｒｉｃ　ｕｎｓａｔｕｒａｔｅｄ　ｆａｔｔｙ　ａｃｉｄｓ．　Ｌｉｐｉｄｓ，　２８，　５６１−５６４　（１９９３）．
１９）　Ｆｏｌｃｈ，　Ｊ．，　Ｌｅｅｓ，　Ｍ．，　ａｎｄ　Ｓｌｏａｎｅ−Ｓｔａｎｌｅｙ，　Ｈ．，　Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｔｈｅ　ｉｓｏｌａｔｉｏｎ　ａｎｄ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｏｆ　ｔｏｔａｌ　ｌｉｐｉｄｅｓ　ｆｒｏｍ　ａｎｉｍａｌ　ｔｉｓｓｕｅｓ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２２６，　４９７−５０９　（１９５７）．
２０）　Ｓｈａｎｋｌｉｎ，　Ｊ．，　Ｗｈｉｔｔｌｅ，　Ｅ．，　ａｎｄ　Ｆｏｘ，　Ｂ．　Ｇ．，　Ｅｉｇｈｔ　ｈｉｓｔｉｄｉｎｅ　ｒｅｓｉｄｕｅｓ　ａｒｅ　ｃａｔａｌｙｔｉｃａｌｌｙ　ｅｓｓｅｎｔｉａｌ　ｉｎ　ａ　ｍｅｍｂｒａｎｅ−ａｓｓｏｃｉａｔｅｄ　ｉｒｏｎ　ｅｎｚｙｍｅ，　ｓｔｅａｒｏｙｌ−ＣｏＡ　ｄｅｓａｔｕｒａｓｅ，　ａｎｄ　ａｒｅ　ｃｏｎｓｅｒｖｅｄ　ｉｎ　ａｌｋａｎｅ　ｈｙｄｒｏｘｙｌａｓｅ　ａｎｄ　ｘｙｌｅｎｅｍｏｎｏｏｘｙｇｅｎａｓｅ．　Ｂｉｏｃｈｅｍｉｓｔｒｙ，　３３，　１２７８７−１２７９４　（１９９４）．
２１）　Ｋａｗａｓｈｉｍａ，　Ｙ．，　Ｓｕｚｕｋｉ，　Ｙ．，　ａｎｄ　Ｈａｓｈｉｍｏｔｏ，　Ｙ．，　Ｄｉｅｔａｒｙ　ｃｏｎｔｒｏｌ　ｏｆ　ｔｈｅ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｐａｌｍｉｔｙｌ−ＣｏＡ　ｉｎ　ｒａｔ　ｌｉｖｅｒ　ｍｉｃｒｏｓｏｍｅｓ．　Ｌｉｐｉｄｓ，　１２，　４３４−４３７　（１９７７）．
２２）　Ｎａｋａｇａｗａ，　Ｍ．，　Ｋａｗａｓｈｉｍａ，　Ｙ．，　ａｎｄ　Ｕｃｈｉｙａｍａ，　Ｍ．，　Ｒｅｌａｔｉｏｎｓｈｉｐ　ｂｅｔｗｅｅｎ　ｄｅｓａｔｕｒａｔｉｏｎ　ａｎｄ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｏｆ　ｐａｌｍｉｔｙｌ−ＣｏＡ　ｉｎ　ｒａｔ　ｌｉｖｅｒ　ｍｉｃｒｏｓｏｍｅｓ．　Ｌｉｐｉｄｓ，　１１，　２４１−２４３　（１９７６）．
２３）　Ｔｈｉｅｄｅ，　Ｍ．　Ａ．　ａｎｄ　Ｓｔｒｉｔｔｍａｔｔｅｒ，　Ｐ．，　Ｔｈｅ　ｉｎｄｕｃｔｉｏｎ　ａｎｄ　ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ　ｏｆ　ｒａｔ　ｌｉｖｅｒ　ｓｔｅａｒｙｌ−ＣｏＡ　ｄｅｓａｔｕｒａｓｅ　ｍＲＮＡ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２６０，　１４４５９−１４４６３　（１９８５）．
２４）　ｄｅ　Ｇｏｍｅｚ　Ｄｕｍｍ，　Ｉ．　Ｎ．，　ｄｅ　Ａｌａｎｉｚ，　Ｍ．　Ｊ．，　ａｎｄ　Ｂｒｅｎｎｅｒ，　Ｒ．　Ｒ．，　Ｄａｉｌｙｖａｒｉａｔｉｏｎｓ　ｏｆ　ｔｈｅ　ｂｉｏｓｙｎｔｈｅｓｉｓ　ａｎｄ　ｃｏｍｐｏｓｉｔｉｏｎ　ｏｆ　ｆａｔｔｙ　ａｃｉｄｓ　ｉｎ　ｒａｔｓ　ｆｅｄ　ｏｎ　ｃｏｍｐｌｅｔｅ　ａｎｄ　ｆａｔ−ｆｒｅｅ　ｄｉｅｔｓ．　Ｌｉｐｉｄｓ，　１９，　９１−９５　（１９８４）．
２５）　Ｓｈａｎｋｌｉｎ，　Ｊ．，　Ｗｈｉｔｔｌｅ，　Ｅ．　Ｊ．，　ａｎｄ　Ｆｏｘ，　Ｂ．　Ｇ．，　Ｍｅｍｂｒａｎｅ　ｂｏｕｎｄ　ｄｅｓａｔｕｒａｓｅｓ　ａｎｄ　ｈｙｄｒｏｘｙｌａｓｅｓ：　ｓｔｒｕｃｔｕｒｅ　ｆｕｎｃｔｉｏｎ　ｓｔｕｄｉｅｓ．　Ｉｎ　”Ｐｌａｎｔ　ｌｉｐｉｄ　ｍｅｔａｂｏｌｉｓｍ”，　ｅｄｓ．　Ｋａｄｅｒ，　Ｊ．−Ｃ．　ａｎｄ　Ｍａｚｌｉａｋ，　Ｐ．，　Ｋｌｕｗｅｒ　Ａｃａｄｅｍｉｃ　Ｐｕｂｌｉｓｈｅｒｓ，　Ｎｅｔｈｅｒｌａｎｄｓ　（１９９５）．
２６）　ｖｏｎ　Ｗｅｔｔｓｔｅｉｎ−Ｋｎｏｗｌｅｓ，　Ｐ．，　Ｏｌｓｅｎ，　Ｊ．　Ｇ．，　Ａｒｎｖｉｇ，　Ｋ．，　ａｎｄ　Ｌａｒｓｅｎ，　Ｓ．，　Ｍｏｌｅｃｕｌａｒ　ａｓｐｅｃｔｓ　ｏｆ　β−ｋｅｔｏａｃｙｌ　ｓｙｎｔｈａｓｅ　（ＫＡＳ）　ｃａｔａｌｙｓｉｓ．　Ｉｎ　”Ｒｅｃｅｎｔ　Ａｄｖａｎｃｅｓ　ｉｎ　ｔｈｅ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　ｏｆ　Ｐｌａｎｔ　Ｌｉｐｉｄｓ”，　ｅｄｓ．　Ｈａｒｗｏｏｄ，　Ｊ．　Ｌ．　ａｎｄＱｕｉｎｎ，　Ｐ．　Ｊ．，　Ｐｏｒｔｌａｎｄ　Ｐｒｅｓｓ，　ｐｐ．　６０１−６０７　（２００１）．
２７）　Ｊａｍｅｓ，　Ｄ．　Ｗ．，　Ｊｒ．，　Ｌｉｍ，　Ｅ．，　Ｋｅｌｌｅｒ，　Ｊ．，　Ｐｌｏｏｙ，　Ｉ．，　Ｒａｌｓｔｏｎ，　Ｅ．，　ａｎｄ　Ｄｏｏｎｅｒ，　Ｈ．　Ｋ．，　Ｄｉｒｅｃｔｅｄ　ｔａｇｇｉｎｇ　ｏｆ　ｔｈｅ　Ａｒａｂｉｄｏｐｓｉｓ　ＦＡＴＴＹ　ＡＣＩＤ　ＥＬＯＮＧＡＴＩＯＮ１　（ＦＡＥ１）　ｇｅｎｅ　ｗｉｔｈ　ｔｈｅ　ｍａｉｚｅ　ｔｒａｎｓｐｏｓｏｎ　ａｃｔｉｖａｔｏｒ．　Ｐｌａｎｔ　Ｃｅｌｌ，　７，　３０９−３１９　（１９９５）．
２８）　Ｌｅｆｋｏｗｉｔｈ，　Ｊ．　Ｂ．，　Ａｃｃｅｌｅｒａｔｅｄ　ｅｓｓｅｎｔｉａｌ　ｆａｔｔｙ　ａｃｉｄ　ｄｅｆｉｃｉｅｎｃｙ　ｂｙ　ｄｅｌｔａ　９　ｄｅｓａｔｕｒａｓｅ　ｉｎｄｕｃｔｉｏｎ：　ｄｉｓｓｏｃｉａｔｉｏｎ　ｂｅｔｗｅｅｎ　ｔｈｅ　ｅｆｆｅｃｔｓ　ｏｎ　ｌｉｖｅｒ　ａｎｄ　ｏｔｈｅｒ　ｔｉｓｓｕｅｓ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ，　１０４４，　１３−１９　（１９９０）．
２９）　Ｓｕｎｅｊａ，　Ｓ．　Ｋ．，　Ｏｓｅｉ，　Ｐ．，　Ｃｏｏｋ，　Ｌ．，　Ｎａｇｉ，　Ｍ．　Ｎ．，　ａｎｄ　Ｃｉｎｔｉ，　Ｄ．　Ｌ．，Ｅｎｚｙｍｅ　ｓｉｔｅ−ｓｐｅｃｉｆｉｃ　ｃｈａｎｇｅｓ　ｉｎ　ｈｅｐａｔｉｃ　ｍｉｃｒｏｓｏｍａｌ　ｆａｔｔｙ　ａｃｉｄ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｉｎ　ｓｔｒｅｐｔｏｚｏｔｏｃｉｎ−ｉｎｄｕｃｅｄ　ｄｉａｂｅｔｉｃ　ｒａｔｓ．　Ｂｉｏｃｈｉｍ．　Ｂｉｏｐｈｙｓ．　Ａｃｔａ，１０４２，　８１−８５　（１９９０）．
３０）　Ｖｏｌｐｅ，　Ｊ．　Ｊ．　ａｎｄ　Ｖａｇｅｌｏｓ，　Ｐ．　Ｒ．，　Ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ｍａｍｍａｌｉａｎ　ｆａｔｔｙ−ａｃｉｄ　ｓｙｎｔｈｅｔａｓｅ．　Ｔｈｅ　ｒｏｌｅｓ　ｏｆ　ｃａｒｂｏｈｙｄｒａｔｅ　ａｎｄ　ｉｎｓｕｌｉｎ．　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　Ｕ　Ｓ　Ａ，　７１，　８８９−８９３　（１９７４）．
３１）　Ｓｈｉｍａｎｏ，　Ｈ．，　Ｙａｈａｇｉ，　Ｎ．，　Ａｍｅｍｉｙａ−Ｋｕｄｏ，　Ｍ．，　Ｈａｓｔｙ，　Ａ．　Ｈ．，　Ｏｓｕｇａ，　Ｊ．，　Ｔａｍｕｒａ，　Ｙ．，　Ｓｈｉｏｎｏｉｒｉ，　Ｆ．，　Ｉｉｚｕｋａ，　Ｙ．，　Ｏｈａｓｈｉ，　Ｋ．，　Ｈａｒａｄａ，　Ｋ．，　Ｇｏｔｏｄａ，　Ｔ．，　Ｉｓｈｉｂａｓｈｉ，　Ｓ．，　ａｎｄ　Ｙａｍａｄａ，　Ｎ．，　Ｓｔｅｒｏｌ　ｒｅｇｕｌａｔｏｒｙ　ｅｌｅｍｅｎｔ−ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ−１　ａｓ　ａ　ｋｅｙ　ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｆａｃｔｏｒ　ｆｏｒ　ｎｕｔｒｉｔｉｏｎａｌ　ｉｎｄｕｃｔｉｏｎ　ｏｆ　ｌｉｐｏｇｅｎｉｃ　ｅｎｚｙｍｅ　ｇｅｎｅｓ．　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２７４，　３５８３２−３５８３９　（１９９９）．
３２）　Ｈｏｒｔｏｎ，　Ｊ．　Ｄ．，　Ｂａｓｈｍａｋｏｖ，　Ｙ．，　Ｓｈｉｍｏｍｕｒａ，　Ｉ．，　ａｎｄ　Ｓｈｉｍａｎｏ，　Ｈ．，　Ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ｓｔｅｒｏｌ　ｒｅｇｕｌａｔｏｒｙ　ｅｌｅｍｅｎｔ　ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎｓ　ｉｎ　ｌｉｖｅｒｓ　ｏｆ　ｆａｓｔｅｄ　ａｎｄ　ｒｅｆｅｄ　ｍｉｃｅ．　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　Ｕ　Ｓ　Ａ，　９５，　５９８７−５９９２　（１９９８）．
３３）　Ｈｏｆｍａｎｎ，　Ｋ．　ａｎｄ　Ｓｔｏｆｆｅｌ，　Ｗ．，　ＴＭｂａｓｅ　−　ａ　ｄａｔａｂａｓｅ　ｏｆ　ｍｅｍｂｒａｎｅ　ｓｐａｎｎｉｎｇ　ｐｒｏｔｅｉｎｓ　ｓｅｇｍｅｎｔｓ．　Ｂｉｏｌ．　Ｃｈｅｍ．　Ｈｏｐｐｅ−Ｓｅｙｌｅｒ，　３４７，　１６６　（１９９３）．
【０１００】
【配列表】

【図面の簡単な説明】
【図１】哺乳類脂肪酸延長酵素のマルチプルアライメントを示す図である。
【図２】ラット延長酵素遺伝子発現の栄養条件的制御を示す写真及びグラフである。
【図３】ラット延長酵素遺伝子発現の組織分布を示す写真である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a technology for synthesizing a fatty acid using a saturated fatty acid-specific chain elongation enzyme.
[0002]
[Prior art]
In the following description, various documents are referred to by Arabic numerals in []. The complete bibliographic details of these references are listed before the sequence listing.
Palmitic acid (C16: 0) and stearic acid (C18: 0) are major components of eukaryotic cell membranes and storage lipids. These saturated fatty acids are supplied by direct nutrient intake and de novo biosynthesis in response to physiological demands [1]. C16: 0 is the main product of fatty acid synthase, which is lengthened to produce C18: 0 and further converted to oleic acid (C18: 1n-9) via a Δ9 desaturation reaction [2] .
[0003]
Vaccenic acid (C18: 1n-7) and other C18: 1 isomers derived from C16: 0 are biosynthesized along different routes, for example, via palmitooleic acid (C16: 1n-7). You. However, in many organisms, they are rather a small number of fatty acid species when compared to C18: 1n-9. Therefore, in such a fatty acid synthesis system, the C16: 0 elongation reaction is considered to be an important step in the biosynthesis of C18 and other long-chain fatty acids.
[0004]
The fatty acid chain elongation system (FACES) has been characterized in mammalian liver and brain [1].
The remarkable activity of FACES is localized in the endoplasmic reticulum (ER) and, to a lesser extent, mitochondria. The vesicle system involves four sequential reactions that begin with the condensation of two substrates, acyl coenzyme A (eg, palmitoyl coenzyme A) and malonyl coenzyme A, which supplies a 2-carbon moiety. After the condensation reaction, reduction of β-ketoacyl coenzyme A, dehydration of β-hydroxyacyl coenzyme A, and reduction of trans-2-enoyl coenzyme A follow. The first reaction, catalyzed by elongase (elongase), is the rate-limiting step of the overall chain elongation reaction [3, 4] and is known to be controlled by diet and hormonal status [ 1].
[0005]
Previous studies have suggested that more than one FACES is present in one cell. Refeeding of laboratory rats with a normal or non-fat diet following fasting resulted in altered prolonged activity of C16: 0 and polyunsaturated fatty acids (PUFAs) in the liver in different ways [5].
[0006]
In addition, in vitro competitive substrate inhibition experiments show that C16: 0 does not affect the elongation of C18: 2n-9 and γ-linolenic acid (C18: 3n-6), whereas these C18 PUFAs Inhibit the elongation reaction of each other. This result indicates the existence of at least two independent FACES. Prasad et al. [6, 7] report that di (2-ethylhexyl) phthalate (a peroxisome proliferator) activates chain extension of saturated fatty acids while suppressing the chain extension reaction of C18: 3n-6 in microsomes. Based on the experimental results, the above concept was supported. Also, different effects of chymotrypsin on the elongation activity of saturated and unsaturated fatty acids have been reported [7].
[0007]
In addition, Luthria and Sprecher [8] have shown that, based on cross-suppression studies, stearidonic acid (C18: 4n-3) and eicosapentaenoic acid (C20: 5n-3) prevent their own chain elongation, but inhibit C22 PUFA chain elongation. It did not hinder. These and other biochemical data suggest that there are at least three enzymes that catalyze the chain extension of mammalian C16: 0 and C18, C20 and C22 PUFAs.
[0008]
To date, there is almost no information on the genes of the above-mentioned elongases. , ELO3 were identified. Recently, mouse genes Ssc1, Ssc2, and Cig30 have been identified that complement yeast strains deficient in each of the ELO1 to ELO3 genes.
[0009]
Tvrdik et al. [9] describe several condensing enzymes involved in the biosynthesis of very long linear fatty acids and sphingolipids in mice. Among these enzymes, Cig30 has been reported to suppress the loss of C20-24 fatty acid synthesis ability in an ELO2-deficient mutant of baker's yeast (Saccharomyces cerevisiae) [10]. In addition, it has been reported that Ssc 1 (a functional homolog of yeast ELO3) [10] is involved in the synthesis of the acyl moiety of sphingolipids.
[0010]
ELO1 was first described as an elongation enzyme required for elongation of myristic acid (C14: 0) [11]. Mutants lacking the gene for the elongase do not require either C16: 0 or C18: 0 for growth because the substrate specificity of the elongase is different from that of the fatty acid synthase.
[0011]
Recently, elongating enzymes that act on C18 PUFAs have been identified in nematodes (Caenorhabditis elegans) [12], fungi (Mortierella alpina) [13], and humans [14].
[0012]
Interestingly, only human elongase (HELO1) can extend C20 PUFAs as well, providing C22 PUFAs that are not present in nematodes and Mortierella fungi. The human elongase could not extend C16: 0 and C18: 0, but catalyzed the extension of C16: 1n-7 to some extent.
[0013]
In the present invention, we disclose a mammalian elongase (designated rELO2 for the rat enzyme). The findings obtained in the present invention provide basic knowledge about the structural and functional differences between multiple FACES in mammals.
[0014]
[Problems to be solved by the invention]
An object of the present invention is to provide a means for extending the chain length of a saturated fatty acid by utilizing an enzyme activity, and in particular, to extend the chain length of a C18: 0 saturated fatty acid to a C20: 0 saturated fatty acid. Is to provide a means for:
[0015]
[Means for Solving the Problems]
In the present invention, the following is provided as means for solving the above-mentioned problems. That is,
A method for synthesizing long-chain fatty acids by a chain-lengthening enzyme,
A transformant is introduced into a suitable host by introducing the nucleotide sequence of SEQ ID NO: 1 or the chain length elongation enzyme gene comprising the nucleotide sequence in which one or several nucleic acids have been deleted, substituted or added to the above sequence, into an appropriate host. The process of creating,
Culturing the transformant to express the chain-lengthening enzyme, and converting the C18: 0 saturated fatty acid to a C20: 0 saturated fatty acid by a saturated fatty acid-specific chain lengthening reaction mediated by the enzyme; Are provided.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
<Enzyme protein>
The enzyme protein used in the present invention is an enzyme protein having the amino acid sequence of SEQ ID NO: 2, and is an enzyme protein (rELO2) that extends the chain length of a fatty acid serving as a substrate, particularly a saturated fatty acid. This enzyme acts on a C16: 0 saturated fatty acid as a substrate, more preferably a C18: 0 saturated fatty acid, and converts it into a fatty acid having a chain length extended by 2 carbons, that is, a C18: 0 saturated fatty acid, more preferably It can be converted to C20: 0 saturated fatty acids.
The enzyme protein used in the present invention is, in addition to the enzyme protein (rELO2) having the amino acid sequence of SEQ ID NO: 2 as described above, one or several amino acids are deleted, substituted, or added in the sequence. And a protein functionally equivalent to the enzyme protein.
[0017]
As used herein, the term “functionally equivalent protein” refers to a protein having an activity of extending the chain length of a fatty acid by two carbon units and acting as a substrate for the fatty acid, preferably C16: 0, by the action of the enzyme protein. It means an enzyme protein having a function of converting a fatty acid, more preferably a C18: 0 saturated fatty acid, into a fatty acid having a 2-carbon extension, preferably a C18: 0 saturated fatty acid, more preferably a C20: 0 saturated fatty acid.
[0018]
<Gene>
In the present invention, a gene encoding the above enzyme protein is transformed into an appropriate host, and the enzyme protein expressed in the host is used. Here, the gene used for transformation has, for example, the nucleotide sequence of SEQ ID NO: 1.
[0019]
Further, in the present invention, in addition to the gene having the nucleotide sequence of SEQ ID NO: 1 as described above, the enzyme comprises a nucleotide sequence in which one or several nucleic acids have been deleted, substituted, or added, A gene encoding a protein functionally equivalent to the protein may be used.
[0020]
The gene can be produced, for example, by a method well-known to those skilled in the art described below. That is,
A reverse transcription reaction is performed based on a primer set designed based on the nucleotide sequence of SEQ ID NO: 1 and mRNA isolated from rat liver, and a PCR method using a prepared cDNA library or the like as a template. Can be amplified. Further, it can be isolated by a method such as hybridization under stringent conditions to a host organism such as Escherichia coli holding the above-mentioned cDNA library using a probe designed based on the above-mentioned nucleotide sequence (the gene of the present invention). See the description of the examples below for a detailed example of a method for isolating.
[0021]
The above-described enzyme protein of the present invention is a translation product of the gene. The enzyme protein can be obtained by incorporating the gene into an appropriate vector, transforming an appropriate host, and then inducing gene expression.
[0022]
<Vector and host>
In the present invention, an appropriate vector is used to transform the gene into a host. The vector used here may be any as long as it can express the gene in a host to which the vector is introduced and transformed, and it is preferable to appropriately select an appropriate vector depending on various conditions such as a host to be used. For example, if the host is yeast, for example, a yeast expression vector pYES2 (Invitrogen, San Diego, USA) that expresses a transgene under the control of the galactose-inducible promoter GAL1 can be used.
[0023]
The host in the present invention is not particularly limited, and an appropriate host is selected depending on the purpose from microorganisms such as Labyrinthuras, bacteria such as Escherichia coli, mammalian cells, insect cells, and the like, and is preferably yeast. . As the yeast, for example, an INVSc1 strain (α / a, his3 / his3, leu2 / leu2, trp1 / trp1, ura3 / ura3; which can be purchased from Invitrogen), which is a yeast (S. cerevisiae) strain, can be used. it can.
[0024]
It is preferable to select an appropriate transformation method depending on the vector and the host used. Those skilled in the art can select an appropriate transformation method from various vector introduction methods such as an electroporation method and a DEAE dextran method.
[0025]
<Method of converting C18: 0 saturated fatty acids to C20: 0 saturated fatty acids>
According to the present invention, chain length of the substrate can be extended by culturing the transformant. In this method, the transformant may be cultured under culture conditions suitable for the host under conditions in which the gene is expressed. At this time, a fatty acid serving as a substrate may be added to the medium at an appropriate concentration. Those skilled in the art can culture the transformant and appropriately select conditions for expressing the gene.
[0026]
For example, when a yeast transformed using the pYES2 vector is used, the method can be performed by culturing under a suitable culture condition for culturing a general yeast. For example, 4% raffinose, In a medium containing 0.7% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, USA), 1% tergitol-type NP-40, 20 μg / ml histidine, 60 μg / ml leucine, and 40 μg / ml tryptophan Culture can be performed under a temperature condition of about 28 ° C. In this case, the expression of the transgene can be induced by adding about 2% (w / v) of galactose and adding about 30 hours of additional culture. If desired, a fatty acid substrate may be added to the medium at a final concentration of about 0.1 mM to 0.5 mM.
[0027]
For separation and analysis of the fatty acid produced by the transformant, a method usually used by those skilled in the art can be used, and for example, analysis can be performed by the following method. That is,
The tissue or cells of the transformant are collected, washed with deionized water, and the lipid fraction is extracted with a mixture of chloroform / methanol (2: 1, v / v). The total lipid fraction is then treated with 10% methanolic hydrochloride (Tokyo Chemical, Tokyo, Japan). The fatty acids methylated in the previous step are extracted into hexane and applied to a gas-liquid chromatograph (GC, model GC-17A, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector for separation.
[0028]
When separating unsaturated fatty acid isomers, chromatography using a column capable of separating unsaturated fatty acid isomers having double bonds at different positions, for example, a TC-70 capillary column (GL Science, Tokyo, Japan) or the like Can be separated by
[0029]
The generated fatty acid methyl ester was identified by sorting using the peak of the fatty acid methyl ester standard solution as an index, and electron ionization using a GC-connected mass spectrometer (GC-MS, GC Mate, JEOL, Tokyo, Japan). It can be implemented by a method such as comparative analysis of mass spectra.
[0030]
That is, the fatty acid methyl ester is mixed with 2-amino-2-methyl 1-propanol and heated at about 180 ° C. for about 18 hours. Next, extraction with dichloromethane and washing with deionized water, a standard fatty acid as a control is treated under the same conditions as in the method for the fatty acid methyl ester, and both are subjected to GC-MS (gas-liquid chromatography-mass spectrum analysis). ). If the standard fatty acid to be compared is not available, a 4,4-dimethyloxazoline (DMOX, 4,4-dimethyloxazole) derivative can be prepared to identify the position of the double bond in the acyl chain.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[0032]
<Materials and methods>
[material]
General chemicals were purchased from Sigma (St. Louis, USA), Katayama Chemical (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). As reagents and restriction enzymes for gene manipulation, those purchased from Takara Shuzo (Kyoto, Japan) and Toyobo (Osaka, Japan) were used.
[0033]
[CDNA cloning]
For cloning of the rELO2 gene, the C. elegans elongase gene sequence [12] was searched for the rat / mouse EST (expressed sequence tag) database in DNA DataBank of Japan (DDBJ) by the BLAST algorithm. Used as a query array.
[0034]
As a result of the above search, mouse EST (accession number AW475681) was searched as a gene having partial homology to the above gene of C. elegans. Met. Another search for a human homolog of the gene on the database found one cDNA sequence (AK027031) that was very similar to the mouse EST sequence. The sequence of the 3 'region deleted in the mouse gene was determined using the rat liver cDNA library (rat derived from Sprague-Dawley strain, Takara Shuzo) as a template, and the oligonucleotide primer FP1 of SEQ ID NO: 3 (5'-ACACGATATTCATCATTCTGGAGAAA-3'). ) And the oligonucleotide primer A145 (5′-TTATCATGTCTGGATCCCTCGAGGGATCCG-3 ′) described in SEQ ID NO: 4 and PCR using ExTaq polymerase (Takara Shuzo). Here, the latter primer corresponds to a part of the sequence on the plasmid pAP2neo used for library construction. The PCR product was re-amplified using rB207F of SEQ ID NO: 5 (5'-ACTGTGCTCCTGTACTCTTGGTACTCCTA-3 '), which is an internal primer derived from the sequences of A145 and AW475568.
[0035]
This PCR resulted in a 2089 bp product encoding the C-terminal portion of the target protein with a TAG stop codon and a 1735-bp long untranslated region. The cDNA fragment containing the rELO2 ORF (open reading frame) was converted into ratEloF1 of SEQ ID NO: 6 (5′-ATCAAAGCTTATGAACATGTCAGTGTGGACTTTTAC-3 ′), which is a primer having recognition sites for Hind III and Xba I, and ratEloR1 of SEQ ID NO: 7, respectively. It was obtained by amplification from a rat cDNA library by PCR using (5'-GCTCTAGACTACTCGGCCTTCGTCGCTTCTT-3 ') and KOD polymerase (TOYOBO).
[0036]
A mouse EST having homology to the HELO1 gene was searched on DDBJ by the same method as described above. As a result, two EST clones, AU079897 and BE535118, were searched as candidates and were found to be ESTs encoding the start and stop codon of the HELO1 homologous gene, respectively. The primers HELOF1 of SEQ ID NO: 8 (5′-AGGGTAAGCTTATGGAACATTTCGATGGCGTCACT-3 ′) and HELOR1 of SEQ ID NO: 9 (5′-CTCTCTAGATCAATCCTTCTCGCTCT which generate Hind III and Xba I sites (italics), respectively. It was designed based on the EST sequence and used to obtain rELO1 (rat homolog of yeast ELO1) cDNA from rat liver cDNA library.
[0037]
A more detailed isolation procedure for the rELO2 gene is shown below.
<< Isolation of conserved region of rELO2 gene >>
Based on the amino acid sequence of the nematode chain elongase F56H11.4, 20 amino acids of SEQ ID NO: 10 (DTIFLVLRKRPLMFLHWYHH), which is considered to be a conserved region, were searched against the mouse EST database (NCBI). As a result, six types of EST sequences were found. Of the six EST sequences, AW475681, AW611298, AI595258, AI428130, AI225632, and AA791133, the latter four showed high homology with the mouse Ssc2 gene. Based on the sequence of AW475681 which had the highest homology to F56H11.4, rAW475FP1 (5′-ACACGAATATTCATCATTCTCTGAGGAA-3 ′) of SEQ ID NO: 11 and rAW475RP1 (5′-TAGTCAGAGTAGCATGCAGCAT) of SEQ ID NO: 12, which are two kinds of primers. 3 ′) was designed to sandwich the storage area. Using these primers, PCR was performed as follows.
[0038]

PCR program
(1) 95 ° C for 2 minutes
(2) 95 ° C for 30 seconds
(3) 55 ° C for 30 seconds
(4) 72 ° C for 1 minute 35 cycles of (2) to (4)
▲ 5 ▼ 72 ℃ 10 minutes
The sequence of the 165 bp gene fragment amplified by this PCR was determined, and the following primers for nested PCR were prepared.
[0039]
RB207F of SEQ ID NO: 5: 5'-ACTGTGCTCCTGTACTCTTGGTACTCCTA-3 '
RB207R of SEQ ID NO: 13: 5'-CATGAACCAACCACCCCCAGCTACCATGT-3 '
<<5'-RACE PCR >>
Rat cDNA library, river (Takara) has a cDNA cloned in a certain direction into a pAPneo3 vector. From the sequences on the pAPneo3 vector, three types of PCR primers A143 (5'-CTTAGGACGCGTAATACGACTCACTATAGGG-3 ') of SEQ ID NO: 14, A145 of SEQ ID NO: 4 (5'-TTATCATGTTCTGGATCCCCGGAGGATCCG-3'), and A146 of SEQ ID NO: 15 ( 5′-TTTTCACTGCATTCTAGTTGTTGGTTTGTCC-3 ′) was designed. Of these, A143 corresponds to the 5 'upstream sequence of the cDNA cloned in the vector, and was used for performing 5' RACE PCR. 5'-RACE PCR was attempted under various conditions using the A143 primer, rAW475RP1 primer and rB207R primer, but the target gene fragment could not be isolated.
[0040]
<<3'-RACE PCR >>
In order to obtain an unacquired portion on the 3 ′ side, 3′-RACE PCR was performed under the following conditions. In addition, the primer A145 corresponds to the sequence on the 3 'upstream side of the cDNA cloned in the pAPneo3 vector.
[0041]

PCR program
(1) 95 ° C for 5 minutes
(2) 95 ° C for 1 minute
(3) 55 ° C for 1 minute
(4) 72 ° C for 3 minutes 35 cycles of (2) to (4)
▲ 5 ▼ 72 ℃ 10 minutes
As a result of analysis by agarose gel electrophoresis, it was confirmed that the DNA fragment considered to be the target portion was amplified. Therefore, nested PCR was further performed using the PCR reaction solution as a template.
[0042]

PCR program
(1) 95 ° C for 5 minutes
(2) 95 ° C for 1 minute
(3) 55 ° C for 1 minute
(4) 72 ° C for 3 minutes 35 cycles of (2) to (4)
▲ 5 ▼ 72 ℃ 10 minutes
Since a DNA fragment of about 2300 bp was amplified by this PCR, sequence analysis was performed, and it was confirmed that the fragment was the target fragment.
[0043]
<< Isolation of rELO2 full length gene >>
Primer ratEloF1 (5′-ATCAaactctATGAACATGTCAGTGTGGACTTTTAC-3 ′; lower case part is Hind III site) designed based on the sequence of mouse EST clone AW475681 and containing the initiation codon of SEQ ID NO: 6 and obtained by 3′-RACE PCR PCR using the primer ratEloR1 (5′-GCTtctagaCTACTCGGCCTTCGTCGCTTCTC-3 ′; lowercase part is XbaI site) designed from the sequence of the fragment containing the stop codon described in SEQ ID NO: 7 was performed as follows. In order to obtain high fidelity, KOD polymerase was used.
[0044]

PCR program
(1) 94 ° C for 2 minutes
(2) 94 ° C for 15 seconds
(3) 60 ° C for 30 seconds
(4) 68 ° C for 1 minute 35 cycles of (2) to (4)
As a result of sequence analysis of the gene fragment of about 850 bp obtained by this PCR, it was found that the gene was the target gene.
[0045]
<< Sequencing around the start codon of rELO2 >>
Since the primer ratEloF1 used for full-length isolation of rELO2 is derived from a mouse, it is necessary to determine the sequence of the rat gene for this portion. Therefore, based on the sequence of AW4755681, primer 5AW475F1 (5′-GCCTTTCATTTCCTCCTACGG-3 ′) described in SEQ ID NO: 16 corresponding to a sequence 5 ′ further upstream from the ratEloF1 portion (about 110 bp upstream from the sequence corresponding to the initiation codon) is designed. Then, PCR was performed as follows.
[0046]

PCR program
(1) 95 ° C for 2 minutes
(2) 95 ° C for 1 minute
(3) 55 ° C for 1 minute
(4) 72 ° C for 1 minute 35 cycles of (2) to (4)
▲ 5 ▼ 72 ℃ 10 minutes
Since amplification of a DNA fragment of about 600 bp was confirmed by this PCR, sequence analysis confirmed that the gene was the target gene. Scrutiny of the nucleotide sequence around the start codon revealed that the 17th G from A of the start codon ATG in the ratEloF1 primer sequence prepared based on the mouse sequence was T in rats. Therefore, in the rat, the leucine at amino acid No. 6 in the amino acid sequence of SEQ ID NO: 2 is tryptophan at this site in the mouse.
[0047]
[Gene expression in yeast]
The PCR products encoding rELO1 and rELO2, respectively, are treated with Hind III and Xba I to correspond to the yeast expression vector pYES2 (Invitrogen, San Diego, USA) which expresses the transgene under the control of the galactose-inducible promoter GAL1. Ligation was performed at the restriction enzyme recognition site. The sequences of all inserted DNAs were analyzed using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and a Genetic Analyzer (type 310, Applied Biosystems, Foster City, Foster City, Foster City). Analyzed using sequencing.
[0048]
The above plasmid was used as the yeast (S. cerevisiae) strain INVSc1 (α / a, his3 / his3, leu2 / leu2, trp1 / trp1, ura3 / ura3; purchased from Invitrogen) or OLE1 (Δ9 desaturation). Enzyme) deficient mutant strain L8-14C (a, ole1Δ :: LEU2, ura3, his4, provided by CE Martin of Rutgers University) [15].
[0049]
Yeast transformants were selected on media plates lacking uracil. During the initial culture of transformants from ole1-deficient mutants that require unsaturated fatty acids for selection, maintenance, and growth, 0.5 mM of C16: 1n-7 and C18: 1n-9 were added to the medium, respectively. The yeast transformants were 4% raffinose, 0.7% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, USA), 1% tergitol type NP-40, 20 μg / ml histidine, 60 μg / ml leucine, and And cultured at 28 ° C. overnight in 50 ml of a medium containing 40 μg / ml tryptophan. The two components mentioned last were not used in the ole1-deficient strain.
[0050]
And ~ 5x10 ⁶ Immediately before reaching a cell density of cells / ml, a fatty acid substrate was added to the culture at a final concentration of 0.1 mM (0.5 mM for ole1 deficient strain). Transgene expression was performed by addition of galactose to 2% (w / v) and further culturing for up to about 30 hours. In lines with a genetic background of ole1 deficiency, the induction period was set to a maximum of 10 hours, since overexpression of the gene would cause growth inhibition of the host [15].
[0051]
[Fatty acid analysis]
Yeast cells were harvested, washed with deionized water, and the lipid fraction was extracted with a chloroform / methanol mixture (2: 1, v / v) as previously described [16].
The total lipid fraction was treated with 10% methanolic hydrochloride (Tokyo Kasei, Tokyo, Japan). The methylated fatty acids were extracted into hexane and applied to a gas-liquid chromatograph (GC, model GC-17A, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. Chromatography was carried out on a TC-70 capillary column (GL Science, Tokyo, Japan) capable of separating unsaturated fatty acid isomers having double bonds at different positions using a temperature rising method (190 ° C./min at 190 ° C./min). went.
[0052]
Sorting using the peak of the fatty acid methyl ester standard solution as an index, and electron using GC (GC-MS, GC Mate, JEOL, Tokyo, Japan) linked to mass spectral analysis as described previously [17]. Each peak was identified by comparative analysis of ionization mass spectra. If the standard fatty acid to be compared was not available, a 4,4-dimethyloxazoline (DMOX, 4,4-dimethyloxazole) derivative was used to identify the position of the double bond in the acyl chain [18].
[0053]
The fatty acid methyl ester was mixed briefly with 2-amino-2-methyl 1-propanol and heated at 180 ° C. for 18 hours. After extraction with dichloromethane and washing twice with deionized water, the DMOX derivative was analyzed by GC-MS (gas-liquid chromatography-mass spectrometry) under the same conditions as for the methyl ester.
[0054]
Male rats of the Sprague-Dawley strain (〜160 g, aged) were purchased from Charles River Japan (Yokohama, Japan) and housed individually and given water and food ad libitum for approximately 5 days.
[0055]
After acclimating to the feeding environment, three rats were fasted for 48 hours and calorically calculated to a control diet consisting of 63.9% carbohydrate, 16.9% fat, and 19.2% protein, vitamins and minerals ( AIN-93G, oriental yeast). After 24 hours of feeding, the rats were again fasted for 48 hours. Each rat was fed a non-fat diet in which the soybean oil and casein in the control diet were replaced with corn starch and defatted casein.
[0056]
Thereafter, the rats were sacrificed and the liver and other organs were rapidly removed. Control rats were fed a control diet over the above period, and each organ was excised as described above. Liver total lipids were extracted by Folch et al. [19] and fatty acid composition was analyzed as described above.
[0057]
[RNA probe and Northern blot analysis]
A DNA fragment containing the cDNAs of rELO1 (1-367 bp) and rELO2 (1-429 bp) was ligated to the pBluescript SK (+) (Stratagene, La Jolla, USA) vector, and Hind III immediately upstream of the cDNA was ligated. The site was digested and linearized. Run-off antisense transcripts were produced using a fluorescein RNA labeling mixture (Roche Diagnostics, Basel, Switzerland) and SP6 or T7 RNA polymerase in the presence of a linearized DNA template. The RNA probe for rat stearoyl-CoA desaturase 1 (SCD1) contains a region corresponding to 103-1177 bp of its open reading frame. Based on the detection intensity of the β-actin gene (chain length of 513 bp), Northern blot was normalized. The probe was denatured by boiling for 5 minutes immediately before use.
[0058]
Polyadenylated RNA was extracted and purified from rat tissues with a μMACS mRNA isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. The mRNA was analyzed on a 1% agarose / formaldehyde gel and then transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech) using a Turboblotter system (Schleicher & Schuell, Keene, USA). The blot membrane was pre-hybridized in ExpressHyb hybridization buffer (Clontech, Palo Alto, USA) for 1 hour at 70-75 ° C.
[0059]
The blot membrane was hybridized overnight at 70-75 ° C. in the same buffer containing 4 ng / ml denatured fluorescein-labeled RNA probe. After hybridization, blots were detected immunochemically on Fuji RX film using a Gene Images CDP-Star detection module (Amersham Pharmacia Biotech).
[0060]
<Result>
[Cloning of two mammalian genes encoding fatty acid elongase]
The nucleotide sequences of the rELO1 and rELO2 cDNAs contained an ORF encoding an amino acid polypeptide of 299 and 267 residues, respectively. FIG. 1 shows the deduced amino acid sequences aligned with the sequences of other mammalian elongases and elongase-like proteins.
[0061]
In FIG. 1, the amino acid sequences translated based on the cDNA sequences of rELO1 (DDBJ accession number AB071985) and rELO2 (AB071986) were converted to HELO1 (AF231981), a human homolog of rELO2 (hELO2, AK027031), and mouse elongation enzyme. With the putative amino acid sequences of Cig30 (mCig30, U97107) and Ssc1 (mSsc1, AF170907). Residues identical to rELO2 are highlighted in black and white. The preserved histidine box is underlined. The rows dotted above the alignment indicate regions of the rELO2 sequence that are likely to form a strong transmembrane helix by TMPredict algorithm analysis [33].
[0062]
The rELO1 sequence has the highest homology to the sequence of the human elongase HELO1 (93.0% identical), followed by the mouse elongase similar protein Ssc2 (57.4%; sequence not shown), It has homology in the order of elongation enzyme Ssc1 (34.3%) and Cig30 (25.8%).
[0063]
The rELO2 sequence was similar to the sequence encoding a human unknown protein (AK027031) (95.8%) named hELO2 in the present invention, and to mouse Cig30 (42.9%).
[0064]
The homology score between rELO1 and rELO2 was 25.0%. By alignment, a distinctive HXXHH motif (described in SEQ ID NO: 17; all of which is conserved between iron-containing enzymes including fatty acid desaturase [20] and elongase [1] (FIG. 1); (Meaning any amino acid). These sequences lack the GSA motif, which has been proposed to be the NADPH-binding site. Also, the GSA motif was found in the sequence of yeast ELO1, but not in yeast ELO2 or ELO3 [10].
[0065]
The pattern of the hydrophobicity plot of rELO2 polypeptide as well as of rELO1 showed similarity to the pattern of the [9-11] elongase, previously reported and predicted to have five transmembrane domains (FIG. 1). Thus, rELO1 is a rat homolog of HELO1, and rELO2 and its human homolog, hELO2, have been elucidated to be novel members of the fatty acid elongase family.
[0066]
[Functional expression of rELO1 and rELO2 genes in yeast]
In order to investigate and compare the function of the gene presumed to be a rat elongation enzyme, the gene was introduced into yeast (S. cerevisiae INVSc1, wild type) to induce expression. Under conditions without the addition of fatty acids, expression of the rELO1 gene newly produced significant amounts of C18: 1n-7 (Table 1). This result is similar to the phenomenon observed when HELO1 (human homolog of rELO1) was expressed in yeast [14], suggesting that endogenous C16: 1n-7 was extended by 2-carbon units. ing.
[0067]
[Table 1]

[0068]
Similarly, eicosenoic acids (C20: 1n-7 and C20: 1n-9), which are detected in small amounts, are assumed to be extension products derived from C18: 1n-7 and endogenous C18: 1n-9, respectively. it was thought.
[0069]
On the other hand, expression of the rELO2 gene in wild-type yeast produces very small amounts of C18: 1n-7 (4.0%), as opposed to rELO1 expression (Table 1), while C18: 1n-9. The content (71.0%) was significantly increased. Due to the difference in the position of the double bond on these C18: 1 isomers, we can distinguish the origin of the products, ie, as described above, C18: 1n-7 becomes C16: 1n-7 by the action of rELO1. Whereas C18: 1n-9 was converted from C18: 0 via Δ9 desaturation by an endogenous enzyme (OLE1p).
[0070]
The fact that C16: 1n-7 did not increase significantly denies the possibility that endogenous OLE1p activity was increased by rELO2 induction. Thus, our results indicate that the increase in C18: 1n-9 due to rELO2 expression is due to activation of the C16: 0 chain elongation reaction.
[0071]
Further, overexpression of rELO2 showed peaks corresponding to C20: 0 and its Δ9-unsaturated product C20: 1n-11, albeit at a trace level similar to C18: 1n-7. However, C20: 1n-9 or C20: 1n-7 was not detected (Table 1). Thus, all detectable monounsaturated fatty acids have a double bond at the Δ9 position and are derived from the corresponding saturated species.
[0072]
To more clearly demonstrate the effects of rELO1 and rELO2, a series of expression experiments were performed using a yeast mutant (L8-14C). Saturated fatty acids are not desaturated in this mutant because the OLE1 (Δ9 desaturase) gene has been disrupted. Since mutants of the ole1Δ genotype require unsaturated fatty acids for normal growth, 0.5 mM C16: 1n-7 was added to the culture medium for gene induction. The trace production of C18: 1n-7 in the control (pYES2, Table 2) may be due to currently unidentified endogenous elongase activity acting on C16: 1n-7.
[0073]
[Table 2]

[0074]
The lack of detection of C18: 1n-9 proved OLE1p inactivation. Under these conditions, rELO1 expression significantly increased C18: 1n-7 (20.3%), although the C16: 1n-7 content was slightly reduced compared to control (2.9%). (Table 2). On the other hand, rELO2 expression increased C18: 0 and decreased C16: 0, so that the ratio of C18: 0 / C16: 0 in the composition of saturated fatty acids was lower than that of control (1.09) and rELO1 (1.15). It increased significantly to a very high value of 2.67 in comparison. This result confirms the prolonged activity of rELO2 in the direction of saturated fatty acid.
[0075]
C20: 0 was below detectable levels (data not shown), possibly due to the low amount of C18: 0 substrate and the lack of rELO2 activity in the ole1Δ genetic background. There is. Considering the fact that it has a structure similar to the elongase (FIG. 1), the above results indicate that rELO2 is a fatty acid elongase that acts mainly on C16: 0, and at a lower level on C18: 0. We support the conclusion that there is.
[0076]
It was also noted that the C18: 1n-7 content was slightly increased in yeast expressing rELO2 (Tables 1 and 2). This is a result suggesting the broad substrate specificity of rELO2. Since rELO1 also catalyzed the elongation reaction from C16: 1n-7 to C18: 1n-7 (Tables 1 and 2), the substrate ranges of the two elongases were considered to overlap. The possibility was verified using a wild-type yeast transformant carrying the rELO1 or rELO2 gene.
[0077]
As can be seen in the results summarized in Table 3, rELO1 expression showed a high conversion rate for C18: 4n-3 followed by C18: 3n-6. In each of these cases, low level production of C22: 4n-3 and C22: 3n-6, respectively, was observed. This result indicates that the produced fatty acids (C20: 4n-3 and C20: 3n-6) were further extended. Each 2-carbon extended product by the expression of rELO1 contains arachidonic acid (C20: 4n-6) and C20: 5n-3, as well as linoleic acid (C18: 2n-6) and α-linolenic acid (C18: Produced even in the presence of 3n-3).
[0078]
[Table 3]

[0079]
The conversion ratio to dihomo-γ-linolenic acid (C20: 3n-6) and mead acid (C20: 3n-9) was less than 3%. In addition, in the case of rELO2 expression, C18 PUFAs were prolonged to their respective C20 PUFAs, but the prolongation response was low (3-7%) and no further conversion of C20 PUFAs could be observed. Induction of expression of a control yeast carrying pYES2 under the same conditions did not produce any extension products (data not shown).
[0080]
These results suggest that elongation of C18 and C20 PUFAs for long chain PUFAs is catalyzed by rELO1, and that minor levels of C18 unsaturation are also elongated by rELO2. Neither rELO1 nor rELO2 exhibited prolonged activity against C22 PUFA.
[0081]
[Diet control of elongation enzyme gene expression]
In rat liver microsomes, total prolonged activity against C16: 0 (similar to Δ9 desaturation activity) has been reported to be reduced by fasting and rapidly increased to high levels by refeeding on a normal or fat free diet. [5, 21].
[0082]
Since actinomycin D (an RNA transcription inhibitor) suppressed the refeeding effect in the chain elongation reaction [21], the increase in elongation activity may be caused by induction of gene expression. To test this possibility, Northern analysis of liver genes was performed using RNA probes complementary to the rELO1, rELO2 and SCD1 genes. In FIG. 2, (A) shows Northern analysis using liver mRNA extracted from three control (CT) and three FFD rats, respectively, using RNA probes complementary to rELO2, rELO1, SCD1, or β-actin. 0.2 μg / lane). The estimated size of the band is shown as kilobase (kb). (B) is a graph showing numerical values obtained by measuring the detected signal intensity of the band (A) by densitometry. Each value is shown by adding the standard deviation to the average value of the level of each measured value with respect to the measured value of β-actin. "*" Marked significantly higher than the corresponding control (p <0.01). This experiment was repeated three times, all showing similar results. As shown in FIG. 2, the amount of rELO2 and SCD1 transcripts was increased 4-5 fold by two repetitions of the fasting and refeeding schedule (FFD) in rats. In contrast, FFD treatment had little effect on rELO1 expression. This result suggests a difference in the expression control mechanism between the two elongases.
[0083]
As with previous data, FFD treatment of rats had a significant effect on fatty acid composition in the liver. That is, the contents of C16: 1n-7 and C18: 1n-9 were increased 5-fold and 2-fold, respectively, as compared with the control rats (Table 4). This result indicates SCD1 activation as reported [22-24].
[0084]
[Table 4]

[0085]
At this time, the C18: 0 content was slightly reduced (to 56%). However, the amount of C18 fatty acids (C18: 0 and C18: 1n-9) in the de novo pathway increased 1.3-fold. This may be the result of rELO2 induction by FFD processing. On the other hand, the content of the main PUFA of the n-6 and n-3 lines was slightly reduced or unchanged. No variation in PUFA composition was indicative of activation or suppression of rELO1. These data further suggest that the expression of rELO1 and rELO2 activity is regulated differently by FFD treatment.
[0086]
The tissue distribution of gene expression was examined by Northern analysis of FFD rats. In FIG. 3, Northern analysis using mRNA samples (0.2 μg / lane) of each organ of the FFD rat shown in the upper part of the figure was performed using an RNA probe for rELO1, rELO, or β-actin. . This experiment was repeated three times, all with similar results. As a result, it was found that rELO1 was expressed in various tissues, and that it was expressed at the highest level in lung and brain (FIG. 3). The results were similar to previous reports [14] except for testis results. In contrast, rELO2 expression was mainly observed in the liver and slightly in the brain, but in other tissues (FIG. 3) or in all examined tissues of control rats (data not shown). Not observed. It was thought that the expression of rELO2 in hepatocytes could contribute to the rapid synthesis of long-chain fatty acids suppressed by FFD treatment.
[0087]
Recent advances in molecular identification of waxes [9] and fatty acid elongation enzymes involved in the biosynthesis of medium / long chain fatty acids [10-14] have revealed the structural properties of these enzymes. These properties include having a characteristic motif expressed as HXXHH (SEQ ID NO: 17), which motifs include diion-oxo (diion-oxo) such as fatty acid desaturases and hydrocarbon hydroxylases. oxo) It is one recognized in the enzyme [20].
[0088]
The histidine residue of the conserved motif has oxygen ₂ -It is believed that it acts as an Fe-chelating ligand to form Fe-O-Fe clusters that contribute to the electron transfer of the dependent redox reaction [25].
[0089]
Another property of the elongase protein is the presence of five hydrophobic stretches that are predicted to be transmembrane domains. This property is consistent with the finding that fatty acid elongation activity is mainly localized in the microsomal membrane fraction. However, these structural features are characteristic of plant enzymes including C16: 0 and KAS II [26], which catalyze the condensation reaction in the chain extension of longer-chain saturated fatty acids, and FAE1 [27], respectively. Not allowed. This fact suggests the existence of different mechanisms in the reaction scheme of these plant enzymes. This difference in mechanism may be related to structural differences in the group of derivatives of catalysable fatty acid substrates. In fact, plant enzymes utilize fatty acids that bind to acyl carrier proteins or glycerides as substrates, whereas animal (and possibly fungal, including yeast) elongases require a coenzyme A-activated substrate. With respect to the condensing enzymes involved in C18: 0 synthesis, the mammalian elongation enzymes hELO2 and rELO2 identified in the present invention are the first of the latter and have no structural similarity to plant enzymes. .
[0090]
In the present invention, a heterologous yeast host expression system was used to demonstrate the elongation activity of rELO2 by C16: 0. When wild-type yeast was used as a host, overexpression of rELO2 resulted in a significant increase in C18: 1n-9 content (Table 1). Next, in order to exclude the involvement of OLE1p from the experimental system, an expression system of rELO2 was constructed in the ole1-deficient yeast mutant L8-14C, and an increase in C18: 0 was observed by induction of gene expression (Table 2). However, the ratio of C18: 0 to C16: 0 (2.67) in the mutant was not as high as that observed in wild-type hosts (7.32, Table 1). This result seems to be because the culture time after the addition of galactose to the ole1Δ host was set shorter than that of the wild-type host. Due to the instability of the ole1Δ host against overexpression of the gene, limitations on the culture time cannot be avoided [15]. In addition to this, C16: 1n-7, a heavily added minor substrate of rELO2, may have become a C16: 0 competitor.
[0091]
Examination of the substrate specificity of rELO1 and rELO2 for PUFA revealed that rELO1 had significantly higher activity to extend C18: 4n-3 and C18: 3n-6 (Table 3). Also, as in previous reports, other PUFAs, including C20: 5n-3, C18: 3n-3, C20: 4n-6 and C18: 2n-6, were prolonged at moderate or low conversion rates. [14].
[0092]
Compared to fatty acids of the same chain length, substrates with higher degrees of unsaturation and substrates with a double bond at the n-3 position tended to be preferentially extended by rELO1. Mammals exhibiting a fatty acid distribution in which the chain length of the major n-3 PUFAs (C20: 5n-3 and C22: 6n-3) is longer than the major n-6 PUFAs (C18: 2n-6 and C20: 4n-6) In animal cells, such high selectivity for n-3 PUFA may partially reflect the content ratio of C18, C20 and C22 PUFA. On the other hand, the substrate specificity of rELO2 catalyzes the conversion of C18 PUFA at low levels, in contrast to the substrate specificity of rELO1.
[0093]
These results emphasize that the substrate selectivity of the elongation enzyme is not so strict, and the specificity differs depending on both the chain length of the acyl group of the substrate and the degree of unsaturation. It should also be noted that rELO1 did not act on saturated fatty acids, a fact which suggests the importance of rELO2 in the C16: 0 elongation reaction. In addition, the fact that none of the elongases exhibited detectable levels of elongation activity on C22 PUFAs suggests the presence of an unidentified elongase.
[0094]
[22] show that both prolongation of C16: 0-coenzyme A in liver microsomes (possibly by rELO2) and desaturation of Δ9 (by SCD1) are activated by fasting and refeeding rats. Is described. SCD1 activation was further enhanced by 2-3 repetitions of a high carb / low fat diet diet schedule [28].
[0095]
In the present invention, we observed that the amount of SCD1 and rELO2 transcripts in hepatocytes was increased 4-5 fold over the respective control under similar conditions (FIG. 2). As with lipids including cholesterol, it has long been known that the activity of these enzymes is regulated by nutritional and hormonal status through changes in blood sugar and insulin levels [1,23,29]. Changes in these nutritional parameters also result in modulation of gene expression of fatty acid synthase that supplies C16: 0 [30].
[0096]
Recent studies have suggested the existence of a regulatory mechanism of SCD1 transcription through activation of sterol regulator binding protein 1 in refeeding following prolonged depletion of dietary lipids [31, 32]. It is quite possible that rELO2 expression is at least partially controlled by intracellular signaling common to factors involved in SCD1 regulation. In contrast to these lipogenic enzymes, FFD treatment of rats did not significantly induce rELO1 expression in hepatocytes (FIG. 2). This result is consistent with previous data that elongation activity on C18: 3n-6 in microsomes was not affected by restricted nutritional status as described above [5].
[0097]
These data suggest the existence of a novel mechanism in the control of the biosynthetic system of de novo fatty acids and PUFAs in cellular homeostasis of saturated and unsaturated fatty acids.
[0098]
【The invention's effect】
As described above in detail, according to the present invention, the chain length of a saturated fatty acid can be extended by utilizing an enzyme activity. More specifically, it is possible to obtain a remarkable effect such as the ability to convert C18: 0 saturated fatty acids into C20: 0 saturated fatty acids via a saturated fatty acid-specific chain lengthening reaction by the enzyme protein of the present invention. it can.
[0099]
[References]
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[0100]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows a multiple alignment of mammalian fatty acid elongation enzymes.
FIG. 2 is a photograph and a graph showing nutritional control of rat elongase gene expression.
FIG. 3 is a photograph showing tissue distribution of rat elongase gene expression.

Claims (2)

A method for synthesizing long-chain fatty acids by a chain-lengthening enzyme,
A transformant is introduced into a suitable host by introducing the nucleotide sequence of SEQ ID NO: 1 or the chain length elongation enzyme gene comprising the nucleotide sequence in which one or several nucleic acids have been deleted, substituted or added to the above sequence, into an appropriate host. The process of creating,
Culturing the transformant to express the chain-lengthening enzyme, and converting the C18: 0 saturated fatty acid into a C20: 0 saturated fatty acid by a saturated fatty acid-specific chain lengthening reaction mediated by the enzyme; A method that includes 2. The method of claim 1, wherein said host is a yeast.

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