JP2000060574A - Apg 12 gene essential to autofuzzy, its detection, manufacture of recombinant protein based on its gene sequence, manufacture of antibody against the same and detection of apg 12 protein using its antibody - Google Patents
Apg 12 gene essential to autofuzzy, its detection, manufacture of recombinant protein based on its gene sequence, manufacture of antibody against the same and detection of apg 12 protein using its antibodyInfo
- Publication number
- JP2000060574A JP2000060574A JP10272435A JP27243598A JP2000060574A JP 2000060574 A JP2000060574 A JP 2000060574A JP 10272435 A JP10272435 A JP 10272435A JP 27243598 A JP27243598 A JP 27243598A JP 2000060574 A JP2000060574 A JP 2000060574A
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- gene
- apg
- apg12
- protein
- autofuzzy
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Links
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明はオートファジーに必
須なＡＰＧ１２遺伝子に関する。TECHNICAL FIELD The present invention relates to an APG12 gene essential for autophagy.
【０００２】[0002]
【従来の技術】細胞は外界からの栄養源が枯渇したとき
に、自己の構成成分の一部を分解して栄養源として再利
用している。これはオートファジーと呼ばれる。このオ
ートフアジーは真核細胞に普遍的な細胞内の分解システ
ムであり、オートファジーは主に非特異的なバルクな分
解システムである。その実体は細胞質の一部を取り囲ん
だオートファゴソームがリソゾーム・液胞と融合すると
いうものである。2. Description of the Related Art When a nutrient source from the outside is exhausted, cells decompose some of their constituent components and reuse them as a nutrient source. This is called autophagy. This autophagy is an intracellular degradation system that is universal to eukaryotic cells, and autophagy is a nonspecific bulk degradation system. Its entity is that the autophagosomes that surround part of the cytoplasm fuse with lysosomes and vacuoles.
【０００３】オートファジーは細胞内の蛋白質、糖、核
酸、オルガネラなどの主に非選択的な分解を担う。この
オートファジーは特に飢餓条件下で顕著に誘導され、こ
れは自己を分解して栄養源として再利用していると考え
られるが、オートファジーは飢餓条件下の細胞の生存維
持のみならず、リモデリング、分化などにも必要と考え
られている。Autophagy is mainly responsible for non-selective degradation of intracellular proteins, sugars, nucleic acids, organelles and the like. This autophagy is significantly induced especially under starvation conditions, which is considered to decompose self and reuse it as a nutrient source. It is considered necessary for modeling and differentiation.
【０００４】このオートファジーという現象は古くより
知られていたが、その分子レベルのメカニズムはほとん
ど明らかにされていなかった。又これまで高等真核生物
ではオートファジーに関する分子は全く同定されていな
かった。This phenomenon of autophagy has been known for a long time, but its molecular level mechanism has hardly been clarified. Until now, no molecule related to autophagy has been identified in higher eukaryotes.
【０００５】[0005]
【発明が解決しようとする課題】そこで我々は鋭意研究
を行なった結果、オートファジーの現象に必須なＡＰＧ
１２遺伝子を発見した。Therefore, as a result of intensive research, we found that APG, which is essential for the phenomenon of autophagy,
I found 12 genes.
【０００６】したがって本発明はこのＡＰＧ１２遺伝子
を提供することを目的とする。又その検出法、その遺伝
子配列に基づくリコンビナント蛋白の作製法、それに対
する抗体の作製法、それに対する抗体を用いたＡｐｇ１
２蛋白の検出法を提供する。Therefore, the present invention aims to provide this APG12 gene. In addition, a method for detecting the same, a method for producing a recombinant protein based on the gene sequence, a method for producing an antibody against it, and an Apg1 using the antibody against it
A method for detecting two proteins is provided.
【０００７】[0007]
【課題を解決するための手段】上記目的を達成するた
め、本発明者達は出芽酵母をモデル系として用い、オー
トファジー不能株（ａｐｇ）を単離することによってそ
の分子機構を探求した。その中の一つとしてＡＰＧ１２
遺伝子をクローニングした結果、この遺伝子が酵母のオ
ートファジーに必須であることを見出した。In order to achieve the above object, the present inventors have investigated the molecular mechanism of Saccharomyces cerevisiae as a model system by isolating a non-autophagy strain (apg). APG12 as one of them
As a result of cloning the gene, it was found that this gene is essential for yeast autophagy.
【０００８】又相補的ＤＮＡあるいはＲＮＡを用いたＡ
ＰＧ１２遺伝子（出芽酵母、ヒト、マウス）の検出法に
関し、ＡＰＧ１２遺伝子の全長あるいは部分に相補的な
ＤＮＡあるいはＲＮＡをラベル（アイソトープ又は非ア
イソトープ）することによって、ＡＰＧ１２ゲノム遺伝
子（サザン法）やＡＰＧ１２のｍＲＮＡ（ノーザン法）
の検出法を見出した。又相補的なオリゴヌクレオチドの
組み合わせによってＰＣＲ法によってＡＰＧ１２遺伝子
を増幅、検出することを見出した。A using complementary DNA or RNA
Regarding the method for detecting the PG12 gene (Budding yeast, human, mouse), by labeling (isotope or non-isotope) DNA or RNA complementary to the full length or part of the APG12 gene, the APG12 genomic gene (Southern method) or APG12 mRNA (Northern method)
The detection method of It was also found that the APG12 gene is amplified and detected by the PCR method by the combination of complementary oligonucleotides.
【０００９】又ＡＰＧ１２遺伝子配列に基づくリコンビ
ナント蛋白の作製に関し、ＡＰＧ１２遺伝子の全長又は
一部、あるいはそれらと他の蛋白質（ＧＳＴ等）との融
合蛋白を、大腸菌などの細胞に発現させることによって
リコンビナント蛋白を作製することを見出した、Regarding the production of a recombinant protein based on the APG12 gene sequence, the recombinant protein is expressed by expressing the full-length or a part of the APG12 gene or a fusion protein thereof with another protein (GST etc.) in cells such as Escherichia coli. Found to make,
【００１０】又リコンビナント蛋白（Ａｐｇ１２蛋白）
の全長又は一部に対する抗体の作製に関し、ＡＰＧ１２
遺伝子配列に基づいたアミノ酸配列全長又は一部を兎、
マウス等に免疫し、ポリクローナル抗体やモノクロナル
抗体を作製することを見出した。Recombinant protein (Apg12 protein)
For the production of antibodies against the full length or a part of APG12
Rabbit with full or partial amino acid sequence based on gene sequence,
It was found that a mouse or the like is immunized to produce a polyclonal antibody or a monoclonal antibody.
【００１１】又リコンビナント蛋白（Ａｐｇ１２蛋白）
に対する抗体を用いたＡｐｇ１２蛋白の検出に関し、Ａ
ｐｇ１２蛋白に対する抗体を用いたウエスタンブロット
法によって、Ａｐｇ１２蛋白の単量体及びＡｐｇ１２蛋
白とＡｐｇ５蛋白の結合体を検出できることを見出し
た。更にこの抗体を用いて、免疫沈降法、ＥＬＩＳＡ法
などによってＡｐｇ１２蛋白を検出できることを見出し
た。Recombinant protein (Apg12 protein)
For the detection of Apg12 protein using an antibody against
It was found that a monomer of Apg12 protein and a conjugate of Apg12 protein and Apg5 protein can be detected by Western blotting using an antibody against pg12 protein. Further, it was found that this antibody can be used to detect Apg12 protein by immunoprecipitation method, ELISA method and the like.
【００１２】本発明は、更に前述したＡＰＧ１２遺伝子
を利用することにより、オートファジーに関連した疾
患、たとえばオートファジーのメカニズムが働かなくな
ることによって分解されるべき蛋白質が蓄積することに
より引き起こさせる疾患、アルツハイマー病や異常蛋白
蓄積病等の診断や治療に応用できるものである。The present invention further utilizes the above-mentioned APG12 gene to cause diseases related to autophagy, for example, diseases caused by the accumulation of proteins to be degraded by the failure of the autophagy mechanism, Alzheimer's disease. It can be applied to diagnosis and treatment of diseases and abnormal protein storage diseases.
【００１３】さらに本発明を説明する。我々発明者達は
出芽酵母をモデル系として用い、オートファジー不能株
（ａｐｇ）を単離することによってその分子機構を探求
した。その中の一つとして、ＡＰＧ１２遺伝子をクロー
ニングした結果この出芽酵母（Ｓａｃｃｈａｒｏｍｙｃ
ｅｓ ｃｅｒｅｖｉｓｉａｅ）のＡＰＧ１２遺伝子（配
列番号１）が、酵母のオートファジーに必須であること
が明らかになった（図１参照）。The present invention will be further described. We have used S. cerevisiae as a model system to explore its molecular mechanism by isolating a non-autophagy strain (apg). As one of them, as a result of cloning the APG12 gene, this budding yeast (Saccharomyc
The es cerevisiae APG12 gene (SEQ ID NO: 1) was revealed to be essential for yeast autophagy (see FIG. 1).
【００１４】又Ａｐｇ１２蛋白はＡｐｇ５蛋白という別
の蛋白質に共有結合するという、ユニークな方法で機能
することを見出した（図２参照）。更に、オートファジ
ーが全ての真核細胞に普遍的であることを考慮して、ヒ
トのＡＰＧ１２に相当する遺伝子（配列番号２）及びマ
ウスのＡＰＧ１２に相当する遺伝子（配列番号３）を発
見した。It was also found that the Apg12 protein functions in a unique way by covalently binding to another protein called Apg5 protein (see FIG. 2). Furthermore, considering that autophagy is ubiquitous in all eukaryotic cells, a gene corresponding to human APG12 (SEQ ID NO: 2) and a gene corresponding to mouse APG12 (SEQ ID NO: 3) were discovered.
【００１５】出芽酵母（ｙＡＰＧ１２）、ヒト（ｈＡＰ
Ｇ１２）、マウス（ｍＡＰＧ１２）のＡｐｇ１２蛋白質
アミノ酸配列の比較が図４に示され、ヒトと出芽酵母で
は２７％のアミノ酸が、ヒトとマウスでは９０％のアミ
ノ酸が一致しており、互いに相同性の高いことが示され
ている。さらにヒトのＡｐｇ１２蛋白もヒトのＡｐｇ５
蛋白と共有結合することが判明し、酵母と同様の機能し
ていることが示された（図３参照）。図５、図６、図７
は、配列番号１、２及び３の配列において、下線は翻訳
開始コドン、二重下線は終止コドンを示すものである。
図８は配列番号１のＡＰＧ１２遺伝子配列より得られた
アミノ酸配列を示し、図９は配列番号２のＡＰＧ１２遺
伝子配列より得られたアミノ酸配列を示し、図１０は配
列番号３のＡＰＧ１２遺伝子配列より得られたアミノ酸
配列を示す図である。Budding yeast (yAPG12), human (hAP
G12) and mouse (mAPG12) Apg12 protein amino acid sequence comparisons are shown in FIG. It has been shown to be high. Furthermore, human Apg12 protein is also related to human Apg5.
It was found to covalently bind to the protein, and it was shown to function similarly to yeast (see FIG. 3). 5, 6, and 7
In the sequences of SEQ ID NOs: 1, 2 and 3, the underline indicates the translation initiation codon and the double underline indicates the stop codon.
8 shows the amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 1, FIG. 9 shows the amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 2, and FIG. 10 shows the amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 3. It is a figure which shows the obtained amino acid sequence.
【００１６】ヒト及びマウスでのＡＰＧ１２遺伝子の発
見は、高等動物でのオートファジーに関連した遺伝子の
初めての発見であり、これまで皆無であったオートファ
ジーの分子レベルでの解析において重要となるものであ
る。The discovery of the APG12 gene in humans and mice is the first discovery of a gene related to autophagy in higher animals, which is important in the analysis of autophagy at the molecular level, which has never existed until now. Is.
【００１７】[0017]
【実施例】つぎに実施例を上げて本発明を説明する。EXAMPLES Next, the present invention will be described with reference to examples.
【００１８】〔実施例１〕 酵母ＡＰＧ１２遺伝子のオ
ートファジーに及ぼす影響
ＡＰＧ１２遺伝子を破壊した株（ｖｅｃｔｏｒ）では窒
素源飢餓状態でもオートファジーは起こらず、ＡＰＧ１
２遺伝子を再び保持させると（ｗｔ）オートファジーの
活性が戻る。よって、配列番号１のＡＰＧ１２遺伝子の
配列がオートファジーに必須な蛋白質をコードしている
ことが示される。さらにＡｐｇ５蛋白との結合に必要な
Ｃ末端のグリシン残基を欠いたＡｐｇ１２蛋白（△Ｇ）
もオートファジー不能いる。Example 1 The yeast APG12 gene
Effect on Autphagy In the strain (vector) in which the APG12 gene was disrupted, autophagy did not occur even under nitrogen starvation, and APG1
When the two genes are retained again (wt), the activity of autophagy is restored. Therefore, it is shown that the sequence of the APG12 gene of SEQ ID NO: 1 encodes a protein essential for autophagy. Furthermore, the Apg12 protein (ΔG) lacking the C-terminal glycine residue necessary for binding to the Apg5 protein
Even autophagy is impossible.
【００１９】〔実施例２〕 酵母Ａｐｇ１２蛋白の発現
と、Ａｐｇ５蛋白との結合
Ａｐｇ１２蛋白に、インフルエンザウイルス由来のＨＡ
エピトープを融合させたものを酵母細胞で発現させた。
Ａｐｇ１２蛋白の単量体と、Ａｐｇ５蛋白との結合体が
存在するのが示される。このことが図２に示されてい
る。[Example 2] Expression of yeast Apg12 protein
And the Apg12 protein bound to the Apg5 protein, HA derived from influenza virus
The fused epitope was expressed in yeast cells.
It is shown that there is a conjugate of the Apg12 protein monomer and the Apg5 protein. This is shown in FIG.
【００２０】〔実施例３〕 ヒトＡｐｇ１２蛋白の発現
と、Ａｐｇ５蛋白との結合
ヒトＡｐｇ１２蛋白に、インフルエンザウイルス由来の
ＨＡエピトープを融合させたものをＣｏｓ−１細胞で発
現させた。Ａｐｇ１２蛋白の単量体（ａ）と、Ａｐｇ５
蛋白との結合体（ｂ、ｃ）とが確認される。ｄ、ｄ’は
分解産物、ｅは修飾されたＡｐｇ１２蛋白を示す。この
ことが図３に示されている。[Example 3] Expression of human Apg12 protein
And binding with Apg5 protein Human Apg12 protein was fused with HA virus-derived HA epitope and expressed in Cos-1 cells. Apg12 protein monomer (a) and Apg5
The bound product (b, c) with the protein is confirmed. d and d ′ are degradation products, and e is a modified Apg12 protein. This is shown in FIG.
【００２１】[0021]
【配列表】ＳＥＱＥＮＣＥ ＬＩＳＴＩＮＧ
〈１１０〉株式会社エルティーティー研究所
〈１２０〉オートファジーに必須なＡＰＧ１２遺伝子、
その検出法、その遺伝子配列に基づくリコンビナント蛋
白の作製法、それに対する抗体の作製法、それに対する
抗体を用いたＡｐｇ１２蛋白の検出法。
〈１３０〉Ｐ−１０４５
〈１６０〉３
〈２１０〉１
〈２１１〉５６１
〈２１２〉ＤＮＡ
〈２１３〉Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉ
ｓｉａｅ
〈４００〉１
〈２１０〉２
〈２１１〉４７３
〈２１２〉ＤＮＡ
〈２１３〉Ｈｕｍａｎ
〈４００〉２
〈２１０〉３
〈２１１〉４４２
〈２１２〉ＤＮＡ
〈２１３〉Ｍｏｕｓｅ
〈４００〉３
[Sequence list] SEQENCE LISTING <110> LTT Research Institute Co., Ltd. <120> APG12 gene essential for autophagy,
A method for detecting the same, a method for producing a recombinant protein based on the gene sequence, a method for producing an antibody against the recombinant protein, and a method for detecting Apg12 protein using the antibody against the recombinant protein. <130> P-1045 <160> 3 <210> 1 <211> 561 <212> DNA <213> Saccharomyces cerevi
siae <400> 1 <210> 2 <211> 473 <212> DNA <213> Human <400> 2 <210> 3 <211> 442 <212> DNA <213> Mouse <400> 3
【図１】酵母ＡＰＧ１２遺伝子のオートファジーに及ぼ
す影響を示す図である。FIG. 1 is a diagram showing the effect of yeast APG12 gene on autophagy.
【図２】酵母Ａｐｇ１２蛋白の発現と、Ａｐｇ５蛋白と
の結合を示す図である。FIG. 2 is a diagram showing expression of yeast Apg12 protein and binding to Apg5 protein.
【図３】ヒトＡｐｇ１２蛋白の発現と、Ａｐｇ５蛋白と
の結合を示す図である。FIG. 3 is a diagram showing expression of human Apg12 protein and binding to Apg5 protein.
【図４】出芽酵母（ｙＡＰＧ１２）、ヒト（ｈＡＰＧ１
２）、マウス（ｍＡＰＧ１２）の蛋白質アミノ酸配列の
比較を示す図である。FIG. 4 Saccharomyces cerevisiae (yAPG12), human (hAPG1)
2) is a diagram showing a comparison of protein amino acid sequences of mouse (mAPG12).
【図５】配列番号１の配列において、下線は翻訳開始コ
ドン、二重下線は終止コドンを示す図である。FIG. 5 is a diagram showing a translation initiation codon and a double underline showing a termination codon in the sequence of SEQ ID NO: 1.
【図６】配列番号２の配列において、下線は翻訳開始コ
ドン、二重下線は終止コドンを示す図である。FIG. 6 is a diagram showing a translation initiation codon and a double underline showing a termination codon in the sequence of SEQ ID NO: 2.
【図７】配列番号３の配列において、下線は翻訳開始コ
ドン、二重下線は終止コドンを示す図である。FIG. 7 is a diagram showing a translation initiation codon and a double underline showing a termination codon in the sequence of SEQ ID NO: 3.
【図８】配列番号１のＡＰＧ１２遺伝子配列より得られ
たアミノ酸配列を示す図である。FIG. 8 is a diagram showing an amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 1.
【図９】配列番号２のＡＰＧ１２遺伝子配列より得られ
たアミノ酸配列を示す図である。FIG. 9 is a diagram showing an amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 2.
【図１０】配列番号３のＡＰＧ１２遺伝子配列より得ら
れたアミノ酸配列を示す図である。FIG. 10 is a diagram showing an amino acid sequence obtained from the APG12 gene sequence of SEQ ID NO: 3.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｒ 1:865） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:865） (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:91） (72)発明者 水島 昇 愛知県岡崎市康生町681サンアメニティキ ャッスル岡崎パークタワー801 Ｆターム(参考） 4B024 AA01 BA80 4B064 AG01 AG27 CA06 CA10 CA19 CC24 DA01 ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI theme code (reference) C12R 1: 865) (C12P 21/02 C12R 1: 865) (C12P 21/02 C12R 1:91) (72) ) Inventor Noboru Mizushima 681 Kousou Town, Okazaki City, Aichi Sun Amenity Castle Okazaki Park Tower 801 F Term (reference) 4B024 AA01 BA80 4B064 AG01 AG27 CA06 CA10 CA19 CC24 DA01
Claims (5)
ァジーに必須なＡＰＧ１２遺伝子。1. An APG12 gene essential for autophagy represented by SEQ ID NOs: 1, 2, and 3.
求項１記載のＡＰＧ１２遺伝子の検出法。2. The method for detecting the APG12 gene according to claim 1, which uses complementary DNA or RNA.
基づくリコンビナント蛋白の作製法。3. A method for producing a recombinant protein based on the APG12 gene sequence according to claim 1.
長または一部に対する抗体の作製法。4. A method for producing an antibody against the full length or a part of the recombinant protein according to claim 3.
する抗体を用いたＡｐｇ１２蛋白の検出法。5. A method for detecting Apg12 protein using an antibody against the recombinant protein according to claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10272435A JP2000060574A (en) | 1998-08-21 | 1998-08-21 | Apg 12 gene essential to autofuzzy, its detection, manufacture of recombinant protein based on its gene sequence, manufacture of antibody against the same and detection of apg 12 protein using its antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10272435A JP2000060574A (en) | 1998-08-21 | 1998-08-21 | Apg 12 gene essential to autofuzzy, its detection, manufacture of recombinant protein based on its gene sequence, manufacture of antibody against the same and detection of apg 12 protein using its antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2000060574A true JP2000060574A (en) | 2000-02-29 |
Family
ID=17513883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10272435A Pending JP2000060574A (en) | 1998-08-21 | 1998-08-21 | Apg 12 gene essential to autofuzzy, its detection, manufacture of recombinant protein based on its gene sequence, manufacture of antibody against the same and detection of apg 12 protein using its antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2000060574A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008099600A (en) * | 2006-10-19 | 2008-05-01 | Juntendo | Transgenic non-human animal |
-
1998
- 1998-08-21 JP JP10272435A patent/JP2000060574A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008099600A (en) * | 2006-10-19 | 2008-05-01 | Juntendo | Transgenic non-human animal |
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