JPWO2005075513A1 - DJ-1 derivatives with oxidized cysteine residues - Google Patents

DJ-1 derivatives with oxidized cysteine residues Download PDF

Info

Publication number
JPWO2005075513A1
JPWO2005075513A1 JP2005517692A JP2005517692A JPWO2005075513A1 JP WO2005075513 A1 JPWO2005075513 A1 JP WO2005075513A1 JP 2005517692 A JP2005517692 A JP 2005517692A JP 2005517692 A JP2005517692 A JP 2005517692A JP WO2005075513 A1 JPWO2005075513 A1 JP WO2005075513A1
Authority
JP
Japan
Prior art keywords
disease
derivative
derivative according
cysteine
oxidative stress
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2005517692A
Other languages
Japanese (ja)
Inventor
朋也 絹見
朋也 絹見
鋭雄 二木
鋭雄 二木
順子 木全
順子 木全
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institute of Advanced Industrial Science and Technology AIST
Original Assignee
National Institute of Advanced Industrial Science and Technology AIST
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute of Advanced Industrial Science and Technology AIST filed Critical National Institute of Advanced Industrial Science and Technology AIST
Publication of JPWO2005075513A1 publication Critical patent/JPWO2005075513A1/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Abstract

本発明は、DJ-1において、１０６位のシステイン残基がシステインスルホン酸に酸化されてなるDJ-1誘導体に関する。該DJ-1誘導体は、神経変性疾患の診断用マーカーとして有用である。The present invention relates to a DJ-1 derivative obtained by oxidizing a cysteine residue at position 106 to cysteine sulfonic acid in DJ-1. The DJ-1 derivative is useful as a diagnostic marker for neurodegenerative diseases.

Description

本発明は、DJ-1の酸化誘導体、該誘導体に対する抗体、並びに、酸化ストレスの指標としてのDJ-1酸化誘導体の利用に関する。   The present invention relates to an oxidized derivative of DJ-1, an antibody against the derivative, and use of the DJ-1 oxidized derivative as an indicator of oxidative stress.

DJ-1はNIH3T3細胞を形質転換させる癌遺伝子として発見された（非特許文献１）。   DJ-1 was discovered as an oncogene that transforms NIH3T3 cells (Non-patent Document 1).

また、パーキンソン病の原因遺伝子としてPARK7が同定され、該遺伝子がDJ-1の点突然変異（166番目のロイシンがプロリンに変異）産物と同一であることが判明した（非特許文献２）。   Moreover, PARK7 was identified as a causative gene of Parkinson's disease, and it was found that the gene was identical to a DJ-1 point mutation (the 166th leucine was mutated to proline) (Non-patent Document 2).

さらに、DJ-1は過酸化水素、パラコートによる酸化ストレスで二次元電気泳動上のスポットがシフトし、スポットの位置の変化からシステインの酸化によることが示唆されている（非特許文献３）。   Furthermore, it is suggested that DJ-1 is caused by oxidation of cysteine from the change in spot position due to the shift of the spot on two-dimensional electrophoresis due to oxidative stress caused by hydrogen peroxide and paraquat (Non-patent Document 3).

さらに、非特許文献４は、DJ-1のX-線結晶構造解析に関する論文であり、パーキンソン病に関わるL166P変異の導入により二量体構造に変化が起こること、X線照射により106番目のシステインが酸化されてスルフィン酸（ＳＯ2Ｈ）になることを示唆している。Furthermore, Non-Patent Document 4 is a paper on X-ray crystal structure analysis of DJ-1, which shows that a change in the dimer structure occurs due to the introduction of the L166P mutation related to Parkinson's disease, and that the 106th cysteine is caused by X-ray irradiation. Is oxidized to sulfinic acid (SO 2 H).

ところで、酸化ストレスは老化や動脈硬化など重要な疾患の原因と考えられており、これらの疾患の早期マーカーとなる酸化ストレスマーカー分子同定は極めて重要である。   By the way, oxidative stress is considered to be a cause of important diseases such as aging and arteriosclerosis, and identification of an oxidative stress marker molecule that is an early marker of these diseases is extremely important.

しかしながら、現在のところ、酸化ストレスマーカーとして８−オキソグアニジンなどの低分子のマーカーが知られるのみであり、有用な酸化ストレスマーカーは実質的に知られていない。
Nagakubo D, Taira T, Kitaura H, Ikeda M, Tamai K, Iguchi-Ariga SM, Ariga H.J-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.Biochem Biophys Res Commun. (1997) 231, 509-13 Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC, Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. (2003) 299, 256-9. Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanezawa Y.Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat. Free Radic Res. (2001) 35, 301-10. Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA. The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease. Proc Natl Acad Sci U S A. (2003) 100, 9256-61. However, at present, only low molecular markers such as 8-oxoguanidine are known as oxidative stress markers, and useful oxidative stress markers are substantially unknown.
Nagakubo D, Taira T, Kitaura H, Ikeda M, Tamai K, Iguchi-Ariga SM, Ariga HJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.Biochem Biophys Res Commun. (1997) 231, 509- 13 Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC, Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.Science. (2003) 299, 256-9. Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanezawa Y. Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat.Free Radic Res. (2001) 35, 301 -Ten. Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA.The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.Proc Natl Acad Sci US A. (2003) 100, 9256-61.

二次元電気泳動の結果を示す。The result of two-dimensional electrophoresis is shown.

本発明は、生体における酸化ストレスを評価し、ひいては、パーキンソン病、アルツハイマー病などの神経変性疾患の有用な診断マーカーを見出すための技術を提供することを目的とする。   It is an object of the present invention to provide a technique for evaluating oxidative stress in a living body and thus finding useful diagnostic markers for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

本発明者は、上記課題に鑑み検討を重ねた結果、DJ-1誘導体の構造を決定し、それにより生体酸化ストレス或いはそれに基づく疾患の診断に関し、有用な技術を確立した。   As a result of repeated investigations in view of the above problems, the present inventor determined the structure of the DJ-1 derivative, thereby establishing a useful technique for diagnosis of biological oxidative stress or diseases based thereon.

即ち、本発明は、以下の発明に関する。
１． DJ-1において、１０６位のシステイン残基がシステインスルホン酸及び／又はシステインスルフィン酸に酸化されてなるDJ-1誘導体。
２． 項１に記載のDJ-1誘導体に対する抗体。
３． 項１に記載のDJ-1誘導体からなる酸化ストレスマーカー。
４． ヒトまたは哺乳動物由来のサンプルにおいて、
(3)項１に記載のDJ-1誘導体の量を測定する工程；または
(4)項１に記載のDJ-1誘導体とDJ-1の比率を測定する工程
を包含することを特徴とする、ヒトまたは哺乳動物における酸化ストレス状態を評価する方法。
５． 項１または２に記載のDJ-1誘導体からなる神経変性疾患の疾患マーカー。
６． 神経変性疾患がパーキンソン病またはアルツハイマー病である項５に記載のマーカー。
７． ヒト由来のサンプルにおいて、
(1)項１に記載のDJ-1誘導体の量を測定する工程；または
(2)項１に記載のDJ-1誘導体とDJ-1の比率を測定する工程
を包含することを特徴とする、ヒトにおける神経変性疾患の診断方法。
８． 神経変性疾患がパーキンソン病またはアルツハイマー病である項７に記載の方法。That is, the present invention relates to the following inventions.
1. A DJ-1 derivative obtained by oxidizing the cysteine residue at position 106 to cysteine sulfonic acid and / or cysteine sulfinic acid in DJ-1.
2. An antibody against the DJ-1 derivative according to Item 1.
3. An oxidative stress marker comprising the DJ-1 derivative according to Item 1.
4). In samples derived from humans or mammals,
(3) a step of measuring the amount of the DJ-1 derivative according to item 1; or
(4) A method for evaluating an oxidative stress state in a human or mammal, comprising a step of measuring the ratio of the DJ-1 derivative according to item 1 and DJ-1.
5. A disease marker for neurodegenerative diseases comprising the DJ-1 derivative according to Item 1 or 2.
6). Item 6. The marker according to Item 5, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.
7). In human-derived samples,
(1) a step of measuring the amount of the DJ-1 derivative according to item 1; or
(2) A method for diagnosing a neurodegenerative disease in a human, comprising a step of measuring the ratio of the DJ-1 derivative according to item 1 and DJ-1.
8). Item 8. The method according to Item 7, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

本発明によれば、酸化ストレス及びパーキンソン病などの神経変性疾患の指標となるDJ-1の構造を決定したことで、これらの病的状態の程度を容易に評価することができるようになった。   According to the present invention, by determining the structure of DJ-1 which is an index of neurodegenerative diseases such as oxidative stress and Parkinson's disease, the degree of these pathological states can be easily evaluated. .

本明細書において、ヒトDJ-1は、配列番号１に示されるアミノ酸配列を有する蛋白質である。ヒトのDJ-1の場合、DJ-1誘導体は、１０６位のシステイン残基(Cys)がシステインスルホン酸(Cys(SO3H))及び／又はシステインスルフィン酸(Cys(SO2H))に酸化された酸化型誘導体（Cys(SO3H)106DJ-1）および（Cys(SO2H)106DJ-1）を包含する。ヒト以外の哺乳動物の場合、DJ-1の１０６位に対応するシステイン残基が酸化された酸化型DJ-1誘導体を意味する。In this specification, human DJ-1 is a protein having the amino acid sequence shown in SEQ ID NO: 1. In the case of human DJ-1, the DJ-1 derivative has a cysteine residue (Cys) at position 106 replaced with cysteine sulfonic acid (Cys (SO 3 H)) and / or cysteine sulfinic acid (Cys (SO 2 H)). Oxidized oxidized derivatives (Cys (SO 3 H) 106 DJ-1) and (Cys (SO 2 H) 106 DJ-1) are included. In the case of mammals other than humans, it means an oxidized DJ-1 derivative in which the cysteine residue corresponding to position 106 of DJ-1 is oxidized.

以下、DJ-1としてヒトDJ-1を例に取り説明するが、ヒト以外の哺乳動物由来のDJ-1についてもヒトDJ-1についての記載を参考にして、当業者であれば容易に本発明を実施することができる。   In the following, human DJ-1 will be described as an example of DJ-1, but those skilled in the art can easily refer to DJ-1 derived from mammals other than humans by referring to the description of human DJ-1. The invention can be implemented.

DJ-1は、酸化ストレスにより、その１０６位のシステイン残基がシステインスルホン酸、或いはその中間体であるシステインスルフィン酸に酸化されて、酸化型のDJ-1誘導体となる。この酸化は、生体に存在する過酸化水素だけでなく、tert-ブチルヒドロペルオキシドなどの他の過酸化物やＰＬＰＣ−ＯＯＨ（フォスファチジルコリンの過酸化物）などの生体由来の過酸化脂質を用いても進行することが本発明者により確認された。従って、このDJ-1の酸化は生理的条件下で生じているものである。データとしては示さないが、本発明者は、アルツハイマー病などの神経変性疾患の患者及び健常者の血液中に、DJ-1酸化体であるCys(SO3H)106DJ-1が存在すること、このDJ-1酸化体は年齢と共に存在量が増加する傾向にあることを本発明者は確認している。Due to oxidative stress, the cysteine residue at position 106 is oxidized to cysteine sulfonic acid or its intermediate, cysteine sulfinic acid, to form an oxidized DJ-1 derivative. This oxidation is not limited to hydrogen peroxide present in the living body, but also other peroxides such as tert-butyl hydroperoxide or living body lipid peroxides such as PLPC-OOH (peroxide of phosphatidylcholine). It has been confirmed by the present inventor that the process proceeds even when used. Therefore, this oxidation of DJ-1 occurs under physiological conditions. Although not shown as data, the present inventor confirmed that Cys (SO 3 H) 106 DJ-1, which is a DJ-1 oxidant, is present in the blood of patients with neurodegenerative diseases such as Alzheimer's disease and healthy individuals. The present inventors have confirmed that the abundance of this oxidized DJ-1 tends to increase with age.

本発明のDJ-1誘導体を認識する抗体（モノクローナル抗体又はポリクローナル抗体）は、DJ-1誘導体を免疫原として用いてラット、マウス、ウサギ、ヤギなどの適当な哺乳動物を免疫し、該免疫哺乳動物の抗体産生細胞を回収し、必要に応じてモノクローナル抗体またはポリクローナル抗体を産生する細胞をスクリーニングし、得られた免疫細胞と哺乳動物のミエローマ細胞とを融合する等の常法に従い得られる。   The antibody (monoclonal antibody or polyclonal antibody) that recognizes the DJ-1 derivative of the present invention immunizes a suitable mammal such as rat, mouse, rabbit, goat, etc. using the DJ-1 derivative as an immunogen. Animal antibody-producing cells are collected, and if necessary, cells that produce monoclonal antibodies or polyclonal antibodies are screened, and the obtained immune cells and mammalian myeloma cells are fused and obtained according to a conventional method.

該抗体は、１０６位のシステインスルホン酸を認識するものであれば、酸化型DJ-1誘導体を特異的に認識することができる。   The antibody can specifically recognize an oxidized DJ-1 derivative as long as it recognizes cysteine sulfonic acid at position 106.

DJ-1誘導体の量またはDJ-1誘導体とDJ-1の比率を測定するためのヒト由来のサンプルとしては、血液、尿、唾液、リンパ液などが挙げられるが、好ましくは血液である。   Examples of the human-derived sample for measuring the amount of the DJ-1 derivative or the ratio of the DJ-1 derivative to DJ-1 include blood, urine, saliva, lymph, etc., preferably blood.

DJ-1誘導体の量またはDJ-1誘導体とDJ-1の比率を測定は、DJ-1誘導体に対する抗体と必要に応じてDJ-1に対する抗体を用い、ＥＬＩＳＡなどのアッセイにより行うのが好ましいが、二次元電気泳動により行うことも可能である。   The amount of DJ-1 derivative or the ratio of DJ-1 derivative to DJ-1 is preferably measured by an assay such as ELISA using an antibody against DJ-1 derivative and, if necessary, an antibody against DJ-1. It is also possible to carry out by two-dimensional electrophoresis.

本発明において、DJ-1酸化誘導体により酸化ストレスの程度並びに診断が可能な神経変性疾患としては、パーキンソン病、アルツハイマー病、筋萎縮性側索硬化症（ＡＬＳ）、脊髄性筋萎縮症候群（ＳＭＡ）、ハンチントン病、脳血管障害後の痴呆が例示される。   In the present invention, the degree of oxidative stress and diagnosis of neurodegenerative diseases that can be diagnosed with a DJ-1 oxidized derivative include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA). Examples include dementia after Huntington's disease and cerebrovascular disorder.

酸化ストレスの強さおよび神経変性疾患（特にパーキンソン病とアルツハイマー病）の関連については、DJ-1誘導体の量（濃度）或いはDJ-1に対する酸化型のDJ-1誘導体の比率が高いほど、酸化ストレスが強く、神経変性疾患に罹っているか、罹る可能性が高いと考えられる。   Regarding the relationship between the intensity of oxidative stress and neurodegenerative diseases (especially Parkinson's disease and Alzheimer's disease), the higher the amount (concentration) of DJ-1 derivative or the ratio of oxidized DJ-1 derivative to DJ-1, the more It is thought that stress is strong and suffers from or is likely to suffer from a neurodegenerative disease.

酸化ストレスの程度を測定する場合、１０６位がシステインスルホン酸及び／又はシステインスルフィン酸に酸化されたDJ-1の量を測定すればよく、これによりパーキンソン病の診断も行うことができる。   When the degree of oxidative stress is measured, the amount of DJ-1 oxidized at position 106 to cysteine sulfonic acid and / or cysteine sulfinic acid may be measured, whereby Parkinson's disease can also be diagnosed.

以下、本発明を実施例に基づきより詳細に説明するが、本発明はこれら実施例には限定されない。
実施例１
（１）試料調製
培養ヒトさい帯静脈血管内皮細胞（Human Umbilical Vein Endothelial Cell: HUVEC, 三光純薬より初代培養品を購入）を直径６ｃｍのディッシュで培養した。培地にEGM-2（三光純薬製）を用い、37oC, 5% CO2のCO2インキュベータにより培養を行う。細胞は４代継代して８０−９０％コンフルエントになった時点で、培地中に88.2 mM過酸化水素を3.4μl添加し（最終濃度100 μ M）１時間CO2インキュベータ内で培養を続けた。過酸化水素処理を行った細胞は回収した後リン酸緩衝液で洗浄し二次元電気泳動に供した。
（２）二次元電気泳動
回収した細胞は等電点電気泳動用溶液（9M尿素、2% CHAPS、65mM DTE、0.5% IPG Buffer(AmershamBioscience社)）280 μ lに溶解し、15000 rpm 20min遠心して不溶成分を除き250 μ lを Immobiline Dry Strip (13 cm長、pIレンジ4-7、AmershamBioscience社)に添加、１２時間膨潤させた。膨潤したのちIPGPhor 電気泳動装置(AmershamBioscience社)により500V１時間、1000V１時間、8000V６時間のタイムプログラムにより等電点電気泳動（一次元目）を行った。泳動終了後、等電点電気泳動を行ったゲルは50 mM Tris-HCl(pH 6.8)、6M尿素、30% グリセロール、2% SDS、20mM DTEにより平衡化し、12.5% のSDSポリアクリルアミドゲル電気泳動によって二次元目の電気泳動を行った。二次元電気泳動ゲルはSyproRuby (Molecular Probes社)により蛍光染色してスポットを可視化し、HUVECへの過酸化水素負荷の有無により位置の移動したスポットを切り出した。EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
Example 1
(1) Sample preparation Cultured human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cell: HUVEC, purchased from Sanko Junyaku Co., Ltd.) were cultured in a 6 cm diameter dish. Use EGM-2 (manufactured by Sanko Junyaku Co., Ltd.) as the medium and culture in a CO 2 incubator with 37 ° C, 5% CO 2 When cells reached 80-90% confluence after 4 passages, 3.4 μl of 88.2 mM hydrogen peroxide was added to the medium (final concentration 100 μM) and the culture was continued in a CO 2 incubator for 1 hour. . The cells treated with hydrogen peroxide were collected, washed with a phosphate buffer, and subjected to two-dimensional electrophoresis.
(2) Two-dimensional electrophoresis The collected cells are dissolved in 280 μl of isoelectric focusing solution (9M urea, 2% CHAPS, 65 mM DTE, 0.5% IPG Buffer (Amersham Bioscience)) and centrifuged at 15000 rpm for 20 min. After removing insoluble components, 250 μl was added to Immobiline Dry Strip (13 cm long, pI range 4-7, Amersham Bioscience) and allowed to swell for 12 hours. After swelling, isoelectric focusing (first dimension) was performed with an IPGPhor electrophoresis apparatus (AmershamBioscience) using a time program of 500 V for 1 hour, 1000 V for 1 hour, and 8000 V for 6 hours. After electrophoresis, the gel subjected to isoelectric focusing was equilibrated with 50 mM Tris-HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, 20 mM DTE, and 12.5% SDS polyacrylamide gel electrophoresis The second dimensional electrophoresis was carried out. The two-dimensional electrophoresis gel was fluorescently stained with SyproRuby (Molecular Probes) to visualize the spot, and the spot moved according to the presence or absence of hydrogen peroxide load on HUVEC was cut out.

なお、過酸化水素添加前の試料（コントロール）を同様にして二次元電気泳動を行った。結果を図１に示す。
（３）質量分析によるタンパク質同定と構造解析
過酸化水素負荷に応答を示した（二次元電気泳動上で位置の移動した）スポットは、トリプシンを用いたゲル内消化によりペプチド断片とした。このペプチド混合物はナノスプレーイオン化LC-MS/MSシステムに導入した。（溶媒A：0.1%ギ酸、5% アセトニトリル水溶液；溶媒B：0.1%ギ酸、98%アセトニトリル水溶液；溶媒Aに対しBを5%から65%まで40分の直線グラジエントで2 μ l/minの流速で送液した。カラムは逆相C18カラム（MAGIC C18, MichromBioresources社）を通してイオントラップ型質量分析計（LCQ-DECA、ThermoElectron社）によりdeta dependent scan modeにより測定を行った。）測定データはMASCOTシステム（Matrix Science社）によりペプチド−マスフィンガープリント法、MS/MSデータサーチ法を併用しタンパク質同定を行った。これらのデータベース検索により、天然型DJ-1、ならびにDJ-1誘導体のトリプシン消化フラグメントの６８％、９３％を同定した。The sample (control) before the addition of hydrogen peroxide was subjected to two-dimensional electrophoresis in the same manner. The results are shown in FIG.
(3) Protein identification and structural analysis by mass spectrometry Spots that showed a response to hydrogen peroxide loading (positions moved on two-dimensional electrophoresis) were made into peptide fragments by in-gel digestion with trypsin. This peptide mixture was introduced into a nanospray ionization LC-MS / MS system. (Solvent A: 0.1% formic acid, 5% acetonitrile aqueous solution; Solvent B: 0.1% formic acid, 98% acetonitrile aqueous solution; B for solvent A from 5% to 65% with a linear gradient of 40 minutes for 2 μl / min flow rate The column was measured in deta dependent scan mode with an ion trap mass spectrometer (LCQ-DECA, ThermoElectron) through a reverse phase C18 column (MAGIC C18, MichromBioresources). (Matrix Science) performed protein identification using the peptide-mass fingerprint method and MS / MS data search method in combination. These database searches identified 68% and 93% of the native DJ-1 and trypsin digested fragments of DJ-1 derivatives.

その結果、二次元電気泳動上で酸性側に現れたスポット(DJ-1誘導体)について、LC-MS/MSの測定データから構造決定を行ったところ、DJ-1の106番目のシステイン残基がシステインスルホン酸（Cys-SO3H）に酸化されていることがわかった。As a result, the structure of the spot (DJ-1 derivative) that appeared on the acidic side on two-dimensional electrophoresis was determined from LC-MS / MS measurement data. It was found that it was oxidized to cysteine sulfonic acid (Cys-SO 3 H).

Claims (8)

DJ-1において、１０６位のシステイン残基がシステインスルホン酸及び／又はシステインスルフィン酸に酸化されてなるDJ-1誘導体。 A DJ-1 derivative obtained by oxidizing the cysteine residue at position 106 to cysteine sulfonic acid and / or cysteine sulfinic acid in DJ-1. 請求項１に記載のDJ-1誘導体に対する抗体。 An antibody against the DJ-1 derivative according to claim 1. 請求項１に記載のDJ-1誘導体からなる酸化ストレスマーカー。 An oxidative stress marker comprising the DJ-1 derivative according to claim 1. ヒトまたは哺乳動物由来のサンプルにおいて、
(1)請求項１に記載のDJ-1誘導体の量を測定する工程；または
(2)請求項１に記載のDJ-1誘導体とDJ-1の比率を測定する工程
を包含することを特徴とする、ヒトまたは哺乳動物における酸化ストレス状態を評価する方法。In samples derived from humans or mammals,
(1) a step of measuring the amount of the DJ-1 derivative according to claim 1; or
(2) A method for evaluating an oxidative stress state in a human or mammal, comprising a step of measuring the ratio of the DJ-1 derivative according to claim 1 and DJ-1. 請求項１または２に記載のDJ-1誘導体からなる神経変性疾患の疾患マーカー。 A disease marker for neurodegenerative disease comprising the DJ-1 derivative according to claim 1 or 2. 神経変性疾患がパーキンソン病またはアルツハイマー病である請求項５に記載のマーカー。 The marker according to claim 5, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease. ヒト由来のサンプルにおいて、
(1)請求項１に記載のDJ-1誘導体の量を測定する工程；または
(2)請求項１に記載のDJ-1誘導体とDJ-1の比率を測定する工程
を包含することを特徴とする、ヒトにおける神経変性疾患の診断方法。In human-derived samples,
(1) a step of measuring the amount of the DJ-1 derivative according to claim 1; or
(2) A method for diagnosing a neurodegenerative disease in a human, comprising a step of measuring the ratio of the DJ-1 derivative according to claim 1 to DJ-1. 神経変性疾患がパーキンソン病またはアルツハイマー病である請求項７に記載の方法。 The method according to claim 7, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.
JP2005517692A 2004-02-04 2005-02-02 DJ-1 derivatives with oxidized cysteine residues Pending JPWO2005075513A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP2004027617 2004-02-04
JP2004027617 2004-02-04
JP2004047517 2004-02-24
JP2004047517 2004-02-24
PCT/JP2005/001478 WO2005075513A1 (en) 2004-02-04 2005-02-02 Dj-1 derivative having acidified cysteine residue

Publications (1)

Publication Number Publication Date
JPWO2005075513A1 true JPWO2005075513A1 (en) 2008-01-10

Family

ID=34840136

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2005517692A Pending JPWO2005075513A1 (en) 2004-02-04 2005-02-02 DJ-1 derivatives with oxidized cysteine residues

Country Status (2)

Country Link
JP (1) JPWO2005075513A1 (en)
WO (1) WO2005075513A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007119237A1 (en) * 2006-04-13 2007-10-25 Ramot At Tel Aviv University Ltd. Methods and kits for diagnosing oxidative stress associated diseases
JP4779103B2 (en) * 2006-06-01 2011-09-28 独立行政法人産業技術総合研究所 Method for producing oxidized polypeptide in which sulfur-containing amino acid residue is oxidized
JP2010183843A (en) * 2009-02-10 2010-08-26 National Institute Of Advanced Industrial Science & Technology Antibody against oxidation type polypeptide containing oxidized sulfur-containing amino acid residue
JP5885158B2 (en) * 2012-02-13 2016-03-15 学校法人同志社 Antibodies against polypeptides containing sulfur-containing amino acid residues
WO2014078623A2 (en) * 2012-11-15 2014-05-22 Brandeis University Tethering cysteine residues using cyclic disulfides
JP5775540B2 (en) * 2013-02-22 2015-09-09 株式会社ファンケル Evaluation method of UV stress applied by UV irradiation of skin

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
JPN6009010421; 'Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓｅａｒｃｈ' ２００１， Ｖｏｌ．３５， ｐｐ．３０１-３１０ *
JPN6009010422; 'Ｆｒｅｅ Ｒａｄｉｃａｌ Ｒｅｓｅａｒｃｈ' ２００１， Ｖｏｌ．３５， ｐｐ．８８５-８９３ *
JPN6009010423; 'Ｓｃｉｅｎｃｅ' ２００３， Ｖｏｌ．２９９， ｐｐ．２５６-２５９ *
JPN6009010425; 'Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ' １９８９， Ｖｏｌ．２６４， Ｎｏ．２１， ｐｐ．１２３３０-１２３３８ *
JPN6009034116; 'Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ ｏｆ ＵＳＡ' ２００３， Ｖｏｌ．１００， Ｎｏ．１６， ｐｐ．９２５６-９２６１ *
JPN6009034117; 'Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ' ２００３， Ｖｏｌ．２７８， Ｎｏ．３３， ｐｐ．３１３７２-３１３７９ *
JPN6009034118; 'Ｂｉｏｃｈｅｍｉｃａｌ Ｊｏｕｒｎａｌ' ２００２， Ｖｏｌ．３６６， ｐｐ．７７７-７８５ *
JPN6009034119; 'Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ' ２００３， Ｖｏｌ．２７８， Ｎｏ．３９， ｐｐ．３７１４６-３７１５３ *
JPN6009034120; 'Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ' ２００２， Ｖｏｌ．２７７， Ｎｏ．２２， ｐｐ．１９３９６-１９４０１ *
JPN6012061821; Ｆｒｅｅ Ｒａｄｉｃａｌ Ｂｉｏｌｏｇｙ ＆ Ｍｅｄｉｃｉｎｅ Ｖｏｌ．３０，Ｎｏ．６, 2001, ｐｐ．６２５-６３５

Also Published As

Publication number Publication date
WO2005075513A1 (en) 2005-08-18

Similar Documents

Publication Publication Date Title
JP5281397B2 (en) Diagnosis of brain injury related disorders
JP5775568B2 (en) Diagnosis method
JPWO2005075513A1 (en) DJ-1 derivatives with oxidized cysteine residues
EP2813853A2 (en) Diagnostic Method for Alzheimer's Disease
Ikezono et al. The diagnostic performance of a novel ELISA for human CTP (Cochlin-tomoprotein) to detect perilymph leakage
US5429947A (en) Diagnosing Alzheimer's disease and schizophrenia
Jonesco et al. The CNS-specific proteoglycan, brevican, and its ADAMTS4-cleaved fragment show differential serological levels in Alzheimer’s disease, other types of dementia and non-demented controls: A cross-sectional study
US10041954B2 (en) Biomarker for psychiatric and neurological disorders
Yokoi et al. Identification and validation of SRC and phospho-SRC family proteins in circulating mononuclear cells as novel biomarkers for pancreatic cancer
RU2661022C2 (en) Tropomyosin isoforms related to alzheimer's disease and moderate cognitive impairments
JP4850001B2 (en) New stress biomarkers and their uses
Patel et al. Avoiding false discovery in biomarker research
KR102232200B1 (en) Alzheimer’s disease diagnostic biomarker
JP7073528B2 (en) Biomarkers for classifying allogeneic transplant recipients
EP2904404B1 (en) Method for aiding diagnosis of alzheimer's disease
US7736864B2 (en) Detection of inflammation
KR102245621B1 (en) Lung Cancer Diagnosis
JP2011226882A (en) Urinary system disease marker and antibody the same, and urinary system disease diagnosis kit
EP4166945A1 (en) Novel biomarkers of membranous glomerulonephritis
JP2013515274A (en) Acidic and cysteine-rich secreted (SPARC) protein SRM / MRM assay
KR101989917B1 (en) Method for the diagnosis of rheumatoid arthritis using capillary electrophoresis with laser induced fluorescence
DK1664795T3 (en) DIAGNOSTIC PROCEDURE FOR Brain Injury-Related Disorders
WO2009099228A1 (en) Method for prediction and test of heart failure, and reagent and kit for use in the test
KR20140094180A (en) Composition for pancreatic cancer diagnosis comprising complememt factor b-specific binding polypeptide or antibody
JP6078244B2 (en) Root white rot disease diagnosis kit for Para rubber tree (Hevea brasiliensis), and method for diagnosing root rot disease of Para rubber tree

Legal Events

Date Code Title Description
A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20090310

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20090511

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A821

Effective date: 20090511

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20090707

A02 Decision of refusal

Free format text: JAPANESE INTERMEDIATE CODE: A02

Effective date: 20091020

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A821

Effective date: 20100115

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20120903

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A821

Effective date: 20120903

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20130122

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A821

Effective date: 20130122