JP6309427B2 - Method for measuring the activity of iduronic acid-2-sulfatase - Google Patents
Method for measuring the activity of iduronic acid-2-sulfatase Download PDFInfo
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Description
本発明は，イズロン酸−２−スルファターゼの天然基質であるヘパラン硫酸，デルマタン硫酸等の繰り返し構造を形成する二糖若しくはその類似体を基質として用いて，試料中に含まれるイズロン酸−２−スルファターゼの酵素活性を測定する方法に関し，より詳しくは，当該二糖を，試料中に含まれるイズロン酸−２−スルファターゼと反応させて，イズロン酸−２−スルファターゼによる当該二糖の加水分解によって生じる脱硫酸化物の生成速度を測定することにより，Ｋｍ値及び最大反応速度を測定する方法に関する。 The present invention relates to iduronic acid-2-sulfatase contained in a sample by using, as a substrate, a disaccharide that forms a repetitive structure such as heparan sulfate, dermatan sulfate or the like, which is a natural substrate of iduronic acid-2-sulfatase. More specifically, the desulfurization caused by hydrolysis of the disaccharide by iduronic acid-2-sulfatase is caused by reacting the disaccharide with iduronic acid-2-sulfatase contained in the sample. The present invention relates to a method for measuring a Km value and a maximum reaction rate by measuring a production rate of an oxide.
イズロン酸−２−スルファターゼ（Ｉ２Ｓ）は，ヘパラン硫酸やデルマタン硫酸のようなグリコサミノグリカン（ＧＡＧ）分子内に存在する硫酸エステル結合を加水分解する活性を有するライソゾーム酵素の１つである。ハンター症候群の患者では，イズロン酸−２−スルファターゼ活性が遺伝的に欠損している。この酵素活性の欠損は，ヘパラン硫酸及びデルマタン硫酸の代謝異常を引き起こし，それは次いで肝臓や腎臓のような組織中にそれらの分子の断片の蓄積や，更には尿中へのヘパラン硫酸及びデルマタン硫酸の排泄をも引き起こす。その結果，これらの異常により，骨格の変形及び重症の精神遅滞を含む，ハンター症候群の患者における種々の症状が引き起こされる。 Iduronic acid-2-sulfatase (I2S) is one of lysosomal enzymes having an activity of hydrolyzing sulfate ester bonds existing in glycosaminoglycan (GAG) molecules such as heparan sulfate and dermatan sulfate. Patients with Hunter syndrome are genetically deficient in iduronic acid-2-sulfatase activity. This deficiency in enzyme activity leads to abnormal metabolism of heparan sulfate and dermatan sulfate, which in turn accumulates fragments of these molecules in tissues such as the liver and kidneys, as well as heparan sulfate and dermatan sulfate in the urine. Also causes excretion. As a result, these abnormalities cause a variety of symptoms in patients with Hunter syndrome, including skeletal deformity and severe mental retardation.
ハンター症候群の患者がＩ２Ｓ活性を僅かしか示さないという事実は，１９７０年代に既に知られており，Ｉ２Ｓ遺伝子の異常がこの疾病の原因であると予想されていた。１９９９年には，Ｉ２Ｓをコードするヒト遺伝子が単離され，この疾患に対する原因遺伝子であることが確認された（非特許文献１）。 The fact that Hunter's syndrome patients show little I2S activity was already known in the 1970s, and abnormalities in the I2S gene were expected to be responsible for this disease. In 1999, a human gene encoding I2S was isolated and confirmed to be a causative gene for this disease (Non-patent Document 1).
現在では，ハンター症候群の患者の体内で不足しているＩ２Ｓ活性を補充するために，遺伝子組換え技術で製造した組換えヒトＩ２Ｓ（ｒｈＩ２Ｓ）を有効成分として含有する製剤を静注して投与する，酵素補充療法が行われている。 Currently, in order to replenish the I2S activity that is lacking in the body of Hunter syndrome patients, a pharmaceutical preparation containing recombinant human I2S (rhI2S) produced by genetic recombination technology is administered intravenously. Enzyme replacement therapy is being used.
Ｉ２Ｓの酵素活性の測定法として，合成基質である４−メチルウンベリフェリル硫酸を基質として用いる方法が知られている（特許文献１，２）。しかしながら，遺伝子組換え技術を用いて製造したヒトＩ２Ｓ（ｒｈＩ２Ｓ）は，糖鎖構造等においてヒト生体内に存在するＩ２Ｓと異なるので，かかる合成基質で測定した活性測定値は，Ｉ２Ｓ本来の酵素活性を反映しないおそれがある。 As a method for measuring the enzyme activity of I2S, a method using 4-methylumbelliferyl sulfate, which is a synthetic substrate, as a substrate is known (Patent Documents 1 and 2). However, since human I2S (rhI2S) produced using genetic recombination technology is different from I2S existing in the human body in terms of sugar chain structure, the measured activity measured with such a synthetic substrate is the original enzyme activity of I2S. May not be reflected.
上記背景の下で，本発明の目的は，Ｉ２Ｓの酵素活性の測定法として，生体内における本来のＩ２Ｓの基質に構造が同一若しくは類似の，２位に硫酸基を有するウロン酸と，アミノ糖とが，１，４−グリコシド結合又は１，３−グリコシド結合により結合したものである二糖を基質として用いる測定法を提供することである。 In view of the above background, an object of the present invention is to measure uronic acid having a sulfate group at the 2-position, which is the same as or similar to the original I2S substrate in vivo, and an amino sugar as a method for measuring I2S enzyme activity. Is to provide a measurement method using a disaccharide that is bound by a 1,4-glycoside bond or a 1,3-glycoside bond as a substrate.
上記目的に向けた研究において，本発明者らは，鋭意検討を重ねた結果，組換え技術を用いて製造したｒｈＩ２Ｓの酵素活性が，イズロン酸２硫酸とアセチルグルコサミン６硫酸とが，α１，４−グリコシド結合により結合した二糖の類縁体を基質として用いて，ｒｈＩ２Ｓと当該基質を反応させたときに生じる脱硫酸化物の生成速度を測定することにより測定できることを見出し，本発明を完成した。すなわち，本発明は以下を提供する。
（１）イズロン酸−２−スルファターゼの酵素活性を測定する方法であって，次のステップすなわち，
（ａ）イズロン酸−２−スルファターゼを，２位に硫酸基を有するウロン酸と，アミノ糖とが，１，４−グリコシド結合又は１，３−グリコシド結合により結合したものである二糖若しくはその塩を基質として，該基質と反応させるステップ，
（ｂ）上記ステップ（ａ）の反応により，該２位の硫酸基が脱硫酸化されることにより生じた生成物を，カラムクロマトグラフィーに付して，該基質から分離させるステップと，
（ｃ）上記ステップ（ｂ）のカラムクロマトグラフィーにより該基質から分離された該生成物を検出するステップとからなる，方法。
（２）該二糖が，下記の一般式（Ｉ）〜（ＩＶ）：
で示される群から選択される二糖若しくはその塩である，上記（１）に記載の方法。
（３）該二糖が，下記の化学式（Ｖ）：
（４）該カラムクロマトグラフィーが，疎水性カラムクロマトグラフィー用カラムと順相／親水性相互作用カラムクロマトグラフィー用カラムとを，この順で接続したものが用いられるものである，上記（１）〜（３）のいずれかに記載の方法。
（５）該イズロン酸−２−スルファターゼが，組換えヒトイズロン酸−２−スルファターゼである，上記（１）乃至（４）のいずれかに記載の方法。As a result of intensive studies, the inventors of the present invention have conducted extensive studies. As a result, the enzyme activity of rhI2S produced by using a recombinant technique is determined to be α1,4 when iduronic acid disulfate and
(1) A method for measuring the enzymatic activity of iduronic acid-2-sulfatase, comprising the following steps:
(A) A disaccharide obtained by combining iduronic acid-2-sulfatase with a uronic acid having a sulfate group at the 2-position and an amino sugar by a 1,4-glycoside bond or a 1,3-glycoside bond, or Reacting a salt as a substrate with the substrate;
(B) subjecting the product produced by desulfation of the sulfate group at the 2-position by the reaction of step (a) to column chromatography to separate it from the substrate;
(C) detecting the product separated from the substrate by the column chromatography in the step (b).
(2) The disaccharide is represented by the following general formulas (I) to (IV):
The method according to (1) above, which is a disaccharide or a salt thereof selected from the group represented by:
(3) The disaccharide has the following chemical formula (V):
(4) The column chromatography is one in which a column for hydrophobic column chromatography and a column for normal phase / hydrophilic interaction column chromatography are connected in this order. The method according to any one of (3).
(5) The method according to any one of (1) to (4) above, wherein the iduronic acid-2-sulfatase is recombinant human iduronic acid-2-sulfatase.
Ｉ２Ｓの酵素活性の測定法として，合成基質である４−メチルウンベリフェリル硫酸を基質として用いる方法が知られている。しかしながら，例えば遺伝子組換え技術を用いて製造したヒトＩ２Ｓ（ｒｈＩ２Ｓ）は，糖鎖構造等においてヒト生体内に存在するＩ２Ｓと異なるので，かかる合成基質で測定した活性測定値は，Ｉ２Ｓ本来の酵素活性を反映しないおそれがある。仮に，ｒｈＩ２Ｓが，合成基質を分解するものの，ヘパラン硫酸及びデルマタン硫酸を効率良く分解できないものであった場合，これを酵素補充療法において患者に投与しても，十分な治療効果が得られない結果となる。本発明によれば，ヒト生体内におけるＩ２Ｓ本来の基質と構造が同一又は類似の物質を，基質として使用して酵素活性を測定するので，組換え技術を用いて製造したヒトＩ２Ｓが，酵素補充療法に適したものであるか否かをより的確に評価できる。 As a method for measuring the enzyme activity of I2S, a method using 4-methylumbelliferyl sulfate, which is a synthetic substrate, as a substrate is known. However, human I2S (rhI2S) produced using, for example, a gene recombination technique is different from I2S existing in the human body in terms of sugar chain structure, etc. May not reflect activity. If rhI2S decomposes the synthetic substrate but cannot efficiently decompose heparan sulfate and dermatan sulfate, even if this is administered to a patient in enzyme replacement therapy, a sufficient therapeutic effect cannot be obtained. It becomes. According to the present invention, since the enzyme activity is measured using a substance having the same or similar structure as the original I2S substrate in the human body as the substrate, the human I2S produced using the recombinant technology is used for enzyme supplementation. It can be evaluated more accurately whether it is suitable for therapy.
本発明において，「イズロン酸−２−スルファターゼ（又はＩ２Ｓ）」というときは，特にヒトイズロン酸−２−スルファターゼ（ｈＩ２Ｓ）であるが，これに限らず，牛，馬等の家畜，イヌ，ネコ等の愛玩動物，マウス，ラット等の実験動物を含む哺乳動物のイズロン酸−２−スルファターゼをも含む。また，「イズロン酸−２−スルファターゼ」には，イズロン酸−２−スルファターゼを構成する１つ又は複数のアミノ酸残基を，置換，欠失，付加又は挿入させたヒトイズロン酸−２−スルファターゼの類似物も含む。 In the present invention, the term “iduronic acid-2-sulfatase (or I2S)” is particularly human iduronic acid-2-sulfatase (hI2S), but is not limited thereto, domestic animals such as cattle and horses, dogs, cats, etc. Also includes iduronic acid-2-sulfatase of mammals including laboratory animals such as pets, mice and rats. In addition, “iduronic acid-2-sulfatase” is similar to human iduronic acid-2-sulfatase in which one or more amino acid residues constituting iduronic acid-2-sulfatase are substituted, deleted, added or inserted. Including goods.
イズロン酸−２−スルファターゼは，遺伝子組換え技術を用いて，遺伝子組換えイズロン酸−２−スルファターゼ（ｒＩ２Ｓ）として生産することができる。ｒＩ２Ｓは，イズロン酸−２−スルファターゼを組込んだ発現ベクターを導入した，ＣＨＯ細胞等の哺乳動物細胞，昆虫細胞，酵母等を用いて生産することができる。例えば，ヒトイズロン酸−２−スルファターゼ遺伝子を発現ベクターに組込み，これを用いて哺乳細胞（例えばチャイニーズハムスター卵巣由来のＣＨＯ細胞）を形質転換し，形質転換細胞を培養することにより，生物学的に活性な組換えヒトイズロン酸−２−スルファターゼ（ｒｈＩ２Ｓ）を製造する方法は，当業者に周知である（ＷＯ２０１２／１０１９９８）。組換えヒトイズロン酸−２−スルファターゼ（ｒｈＩ２Ｓ）は，好ましくは５２５個のアミノ酸残基からなり単一の糖鎖ポリペプチドを含んでなるヒト野生型Ｉ２Ｓである組換えタンパク質であるが，野生型ヒトＩ２Ｓと比較して１個又は２個以上のアミノ酸の置換，欠失，付加又は挿入を有するものであるヒト変異型のＩ２Ｓである組換えタンパク質を排除するものではない。 Iduronic acid-2-sulfatase can be produced as genetically modified iduronic acid-2-sulfatase (rI2S) using a gene recombination technique. rI2S can be produced using mammalian cells such as CHO cells, insect cells, yeast, etc., into which an expression vector incorporating iduronic acid-2-sulfatase has been introduced. For example, the human iduronic acid-2-sulfatase gene is incorporated into an expression vector, and this is used to transform mammalian cells (eg, CHO cells derived from Chinese hamster ovary) and culture the transformed cells for biologically active activity. Methods for producing novel recombinant human iduronate-2-sulfatase (rhI2S) are well known to those skilled in the art (WO2012 / 101998). Recombinant human iduronic acid-2-sulfatase (rhI2S) is a recombinant protein that is human wild type I2S, preferably consisting of 525 amino acid residues and comprising a single carbohydrate polypeptide, It does not exclude recombinant proteins that are human variants of I2S that have one, two or more amino acid substitutions, deletions, additions or insertions compared to I2S.
本発明において，酵素活性の測定対象となるべきｒＩ２Ｓは，例えば，ハンター症候群の患者の治療剤として製造されたｒＩ２Ｓである。かかるｒＩ２Ｓは，ハンター症候群の患者体内において，ヘパラン硫酸やデルマタン硫酸のようなグリコサミノグリカン（ＧＡＧ）分子内に存在する硫酸エステル結合を加水分解して薬効を示すので，これら天然基質を加水分解する活性を有するか否かを，製造工程において予め評価する必要がある。なお，本発明において「天然基質」というときは，生体内においてｒＩ２Ｓの酵素活性によって脱硫化される物質と同一若しくは類似の構造を有する物質又はその塩のことをいう。 In the present invention, rI2S to be measured for enzyme activity is, for example, rI2S produced as a therapeutic agent for patients with Hunter syndrome. Such rI2S exhibits a medicinal effect by hydrolyzing sulfate ester bonds existing in glycosaminoglycan (GAG) molecules such as heparan sulfate and dermatan sulfate in the body of a Hunter syndrome patient. It is necessary to evaluate in advance in the manufacturing process whether or not it has the activity to be performed. In the present invention, the term “natural substrate” refers to a substance having the same or similar structure as that of a substance desulfurized in vivo by the enzyme activity of rI2S or a salt thereof.
本発明において，イズロン酸−２−スルファターゼの基質として用いられるものは，２位に硫酸基を有するウロン酸と，アミノ糖とが，１，４−グリコシド結合又は１，３−グリコシド結合により，特に，α１，４−グリコシド結合又はα１，３−グリコシド結合により結合したものである二糖であり，好ましくは，下記の一般式（Ｉ）〜（ＩＶ）で示されるものである。なお，式（Ｉ）〜（ＩＶ）中，Ｒ１は−ＮＨ２，−ＮＨＣＯＣＨ３又は−ＮＨＳＯ３Ｈ；Ｒ２はＯＳＯ３Ｈ；Ｒ３はＣＯＯＨ；Ｒ４はＣＨ２ＯＨ又はＣＨ２ＯＳＯ３Ｈ；Ｒ５はＯＨ又はＯＳＯ３Ｈを，それぞれ独立して示す。ここで，１，４−グリコシド結合というときは，ウロン酸の１位の炭素に結合した水酸基とアミノ糖の４位の炭素に結合した水酸基とが脱水縮合した化学式で示されるものをいい，１，３−グリコシド結合とは，ウロン酸の１位の炭素に結合した水酸基とアミノ糖の３位の炭素に結合した水酸基とが，脱水縮合した化学式で示されるものをいう。In the present invention, what is used as a substrate for iduronic acid-2-sulfatase is a uronic acid having a sulfate group at the 2-position and an amino sugar, particularly by a 1,4-glycoside bond or a 1,3-glycoside bond. , Α1,4-glycosidic bonds or α1,3-glycosidic bonds, preferably disaccharides represented by the following general formulas (I) to (IV). In the formulas (I) to (IV), R1 is —NH 2 , —NHCOCH 3 or —NHSO 3 H; R2 is OSO 3 H; R3 is COOH; R4 is CH 2 OH or CH 2 OSO 3 H; R5 Represents OH or OSO 3 H independently. Here, the 1,4-glycoside bond refers to a chemical formula obtained by dehydration condensation of a hydroxyl group bonded to the 1st carbon of uronic acid and a hydroxyl group bonded to the 4th carbon of amino sugar. 1,3-glycoside bond refers to a compound represented by a chemical formula in which a hydroxyl group bonded to the 1st carbon of uronic acid and a hydroxyl group bonded to the 3rd carbon of an amino sugar are dehydrated and condensed.
下記の一般式（Ｖ）で示される二糖は，本発明において，基質として好適に用いることができる。当該二糖は塩であってもよく，その場合，例えば，Ｒ２がＯＳＯ３Ｎａ，Ｒ３がＣＯＯＮａ，Ｒ４がＣＨ２ＯＳＯ３Ｎａであるナトリウム塩が好適に使用できる。また，当該二糖は，Ｈｅｐａｒｉｎ ｄｉｓａｃｃｈａｒｉｄｅ−Ａ又はＨｅｐａｒｉｎ ｕｎｓａｔｕｒａｔｅｄ ｄｉｓａｃｃｈａｒｉｄｅ Ｉ−Ａの名称で，そのナトリウム塩が市販されている。In the present invention, a disaccharide represented by the following general formula (V) can be suitably used as a substrate. The disaccharide may be a salt. In this case, for example, a sodium salt in which R2 is OSO 3 Na, R3 is COONa, and R4 is CH 2 OSO 3 Na can be preferably used. The disaccharide is commercially available under the name Heparin disaccharide-A or Heparin undissociated disaccharide IA.
上記一般式（Ｉ）〜（Ｖ）で示される糖は，Ｉ２Ｓによってウロン酸の２位の硫酸基が脱硫化される。上記一般式（Ｖ）で示される二糖は，Ｉ２Ｓによって脱硫化されることにより，下記の一般式（ＶＩ）で示される生成物を生じる。この物質は，Ｈｅｐａｒｉｎ ｄｉｓａｃｃｈａｒｉｄｅ ＩＩ−Ａの名称で，そのナトリウム塩が市販されている。 In the sugars represented by the above general formulas (I) to (V), the sulfate group at the 2-position of uronic acid is desulfurized by I2S. The disaccharide represented by the above general formula (V) is desulfurized by I2S to yield a product represented by the following general formula (VI). This material has the name Heparin disaccharide II-A and its sodium salt is commercially available.
本発明において，Ｉ２Ｓの反応速度は，上記一般式（Ｉ）〜（Ｖ）で示される糖が脱硫化されて生じる脱硫化物の，単位時間当たりの生成量又は反応溶液中における濃度の増加量として測定される。 In the present invention, the reaction rate of I2S is expressed as the amount of desulfurized product produced by desulfurization of the sugars represented by the above general formulas (I) to (V) per unit time or the increase in concentration in the reaction solution. Measured.
Ｉ２Ｓの反応速度は，上記の一般式（Ｖ）で示される二糖若しくはその塩を基質として用いた場合，反応終了後の反応溶液を，疎水性カラムクロマトグラフィーと順相／親水性相互作用カラムクロマトグラフィーとに，この順で連続して通過させることにより，上記の一般式（Ｖ）で示される二糖が脱硫化されて生じる上記の一般式（ＶＩ）で示される脱硫化物を，上記の一般式（Ｖ）で示される二糖から分離して定量することにより，単位時間当たりに生成した当該脱硫化物の量として求めることができる。このとき用いられる疎水性カラムクロマトグラフィー用カラムは，好ましくは充填剤としてブチル化シリカゲルを充填したものであり，順相／親水性相互作用クロマトグラフィー用カラムは，好ましくは充填剤としてアミノ基を結合させたシリカゲルを充填したものである。また，このときのカラムクロマトグラフィーの移動相は，好ましくはリン酸二水素ナトリウム水溶液又はリン酸緩衝液である。リン酸二水素ナトリウム水溶液を移動相として用いる場合，その濃度は，好ましくは０．３〜０．５Ｍであり，より好ましくは０．４Ｍである。 When the disaccharide represented by the above general formula (V) or a salt thereof is used as a substrate, the reaction rate of I2S is determined by using hydrophobic column chromatography and normal phase / hydrophilic interaction column. The desulfurized product represented by the above general formula (VI) produced by desulfurization of the disaccharide represented by the above general formula (V) is passed through the chromatography continuously in this order. By separating and quantifying from the disaccharide represented by the general formula (V), it can be determined as the amount of the desulfurized product generated per unit time. The column for hydrophobic column chromatography used here is preferably packed with butylated silica gel as a packing material, and the column for normal phase / hydrophilic interaction chromatography preferably has an amino group bonded as a packing material. It is filled with silica gel. The mobile phase of column chromatography at this time is preferably a sodium dihydrogen phosphate aqueous solution or a phosphate buffer. When an aqueous sodium dihydrogen phosphate solution is used as the mobile phase, the concentration is preferably 0.3 to 0.5M, more preferably 0.4M.
また，Ｉ２Ｓの反応速度は，上記の一般式（Ｖ）で示される二糖若しくはその塩を基質として用いた場合，反応溶液中に含まれるＩ２Ｓの濃度を，好ましくは０．０００１〜０．０２５ｍｇ／ｍＬ，より好ましくは０．０００５〜０．０２ｍｇ／ｍＬ，更に好ましくは０．００１〜０．０１ｍｇ／ｍＬに調整して測定される。また，このとき反応溶液中に含まれる基質の初濃度は，好ましくは０．０２〜３．０ｍＭの範囲であり，より好ましくは０．０２〜１．５ｍＭの範囲であり，更に好ましくは０．１０〜１．２ｍＭの範囲である。また，このときの反応時間は，好ましくは３０秒から１０分間であり，より好ましくは１分から５分間である。 In addition, when the disaccharide represented by the general formula (V) or a salt thereof is used as a substrate, the I2S reaction rate is preferably the concentration of I2S contained in the reaction solution, preferably 0.0001 to 0.025 mg. / ML, more preferably 0.0005 to 0.02 mg / mL, and still more preferably 0.001 to 0.01 mg / mL. At this time, the initial concentration of the substrate contained in the reaction solution is preferably in the range of 0.02 to 3.0 mM, more preferably in the range of 0.02 to 1.5 mM, and still more preferably 0.8. The range is 10 to 1.2 mM. The reaction time at this time is preferably 30 seconds to 10 minutes, more preferably 1 minute to 5 minutes.
Ｉ２Ｓの反応速度は，基質の初濃度（Ｓ０）の逆数に対して，単位時間当たりに生成した脱硫化物の量を反応速度（Ｖ）としてその逆数（１／Ｖ）をプロット（Ｌｉｎｅｗｅａｖｅｒ−Ｂｕｒｋプロット）して回帰分析し，得られた直線のｙ軸との切片を１／Ｖｍａｘ（Ｖｍａｘは酵素反応の最大速度），ｘ軸との切片を−１／Ｋｍ（Ｋｍはミカエリス定数）とし，ＶｍａｘとＫｍとして求めることができる。The reaction rate of I2S is plotted against the reciprocal of the initial concentration (S 0 ) of the substrate, and the reciprocal (1 / V) is plotted (Lineweaver-Burk) with the amount of desulfurized product generated per unit time as the reaction rate (V). (Plot) and regression analysis, and the intercept with the y-axis of the obtained straight line is 1 / Vmax (Vmax is the maximum rate of the enzyme reaction), and the intercept with the x-axis is -1 / Km (Km is the Michaelis constant), Vmax and Km can be obtained.
以下，実施例を参照して本発明を更に詳細に説明するが，本発明が実施例に限定されることは意図しない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, it is not intended that the present invention be limited to the examples.
〔試薬の調製〕
２５０ｍｇのウシ血清アルブミン（ＢＳＡ）及び０．５ｇのＴｒｉｔｏｎ−Ｘ１００を，全量が５００ｍＬとなるように５ｍＭ酢酸緩衝液（ｐＨ４．５）に溶解し，試液除粒子用フィルター（Ｂｏｔｔｌｅ Ｔｏｐ Ｖａｃｕｕｍ Ｆｉｌｔｅｒ，０．２２μｍ ＣＡ Ｍｅｍｂｒａｎｅ，コーニング製）でフィルター濾過したものを，希釈用緩衝液とした。リン酸二水素ナトリウム二水和物６２．４ｇに水を加えて全量を１０００ｍＬにした後，孔径０．２２μｍのメンブレンフィルターを用いてろ過したものを，移動相とした。５ｍｇのＨｅｐａｒｉｎ ｄｉｓａｃｃｈａｒｉｄｅ Ｉ −Ａ（ＨＤ−Ｉ−Ａ，Ｄｅｘｔｒａ Ｌａｂｏｒａｔｏｒｉｅｓ製）に，１ｍＬの希釈用緩衝液を添加して混和したものを基質原液（ＨＤ−Ｉ−Ａの濃度：１６．５２ｍＭ）とした。５ｍｇのＨｅｐａｒｉｎ ｄｉｓａｃｃｈａｒｉｄｅ ＩＩ −Ａ（ＨＤ−ＩＩ−Ａ，Ｄｅｘｔｒａ Ｌａｂｏｒａｔｏｒｉｅｓ製）に，５００μＬの希釈用緩衝液を添加して混和したものを，希釈用緩衝液で２倍希釈したものを標準原液（ＨＤ−ＩＩ−Ａの濃度：１０ｍＭ）とした。５０μＬの標準原液に，２５μＬの希釈用緩衝液と１２５μＬの移動相とを加えて混和し，これを標準溶液とした（ＨＤ−ＩＩ−Ａの濃度：２５ｍＭ）。(Preparation of reagents)
250 mg of bovine serum albumin (BSA) and 0.5 g of Triton-X100 were dissolved in 5 mM acetate buffer (pH 4.5) so that the total amount was 500 mL, and the test solution removal particle filter (Bottom Top Vacuum Filter, 0) (22 μm CA Membrane, manufactured by Corning) was used as a buffer for dilution. Water was added to 62.4 g of sodium dihydrogen phosphate dihydrate to a total volume of 1000 mL, and then filtered using a membrane filter having a pore size of 0.22 μm was used as the mobile phase. A substrate stock solution (HD-IA concentration: 16.52 mM) was prepared by adding 1 mL of dilution buffer to 5 mg of Heparin disaccharide I-A (HD-IA, manufactured by Dexter Laboratories) and mixing. did. A standard stock solution (HD) of 500 mg Heparin disaccharide II-A (HD-II-A, manufactured by Dextra Laboratories) with 500 μL of dilution buffer added and mixed twice with dilution buffer. -Concentration of II-A: 10 mM). To 50 μL of the standard stock solution, 25 μL of dilution buffer and 125 μL of mobile phase were added and mixed to obtain a standard solution (HD-II-A concentration: 25 mM).
〔ｒｈＩ２Ｓ溶液の調製〕
組換え体ヒトイズロン酸−２−スルファターゼ（ｒｈＩ２Ｓ）の精製品を，公知の手法（米国特許公報５７９８２３９，国際公開公報ＷＯ２０１２／１０１９９８）に準じて調製した。ｒｈＩ２Ｓの精製品を含有する溶液（ｒｈＩ２Ｓ溶液）を，純水で３〜５倍希釈した後に，遠心式ろ過ユニット（アミコンウルトラ−４，ミリポア製）を用いて濃縮した。得られた濃縮液を純水で３〜５倍希釈した後に，再度遠心式ろ過ユニットを用いて濃縮した。この希釈−濃縮操作を，元の溶液の純水での総希釈倍率が１万倍に達するまで繰り返して行い，ｒｈＩ２Ｓ溶液を脱塩した。脱塩したｒｈＩ２Ｓ溶液に含まれる蛋白質の濃度を測定し，蛋白質濃度が０．５ｍｇ／ｍＬとなるように純水でｒｈＩ２Ｓ溶液を希釈し，さらに，この希釈溶液を蛋白質濃度が０．０１ｍｇ／ｍＬとなるように希釈用緩衝液で希釈し，次いで，３７℃に加温した。[Preparation of rhI2S solution]
A purified product of recombinant human iduronate-2-sulfatase (rhI2S) was prepared according to a known technique (US Patent Publication No. 5798239, International Publication No. WO2012 / 101998). A solution (rhI2S solution) containing a purified product of rhI2S was diluted 3 to 5 times with pure water, and then concentrated using a centrifugal filtration unit (Amicon Ultra-4, manufactured by Millipore). The obtained concentrated solution was diluted 3 to 5 times with pure water, and then concentrated again using a centrifugal filtration unit. This dilution-concentration operation was repeated until the total dilution ratio of the original solution with pure water reached 10,000 times to desalinate the rhI2S solution. The concentration of the protein contained in the desalted rhI2S solution is measured, the rhI2S solution is diluted with pure water so that the protein concentration becomes 0.5 mg / mL, and this diluted solution is further diluted with a protein concentration of 0.01 mg / mL. Was diluted with a buffer solution for dilution, and then warmed to 37 ° C.
〔酵素反応１〕
基質原液を，ＨＤ−Ｉ−Ａの濃度が２．４８ｍＭとなるように，希釈用緩衝液で希釈して基質溶液とした。基質溶液を５０μＬずつ試験管にとり３７℃に加温し，これに上記の３７℃に加温したｒｈＩ２Ｓ溶液を２５μＬ添加して混和し反応溶液とした。従って，このときの反応溶液中のＨＤ−Ｉ−Ａの初濃度は，１．６５ｍＭであった。次いで，反応溶液を３７℃で反応開始から３分，５分，１０分，３０分及び６０間静置して，ｒｈＩ２ＳによるＨＤ−Ｉ−Ａの脱硫化反応をさせた。次いで，反応溶液を１００℃で５分間加熱して酵素を失活させて反応を停止させた。[Enzyme reaction 1]
The substrate stock solution was diluted with a dilution buffer so that the concentration of HD-IA was 2.48 mM to obtain a substrate solution. 50 μL of the substrate solution was placed in a test tube and heated to 37 ° C., and 25 μL of the above rhI2S solution heated to 37 ° C. was added and mixed to obtain a reaction solution. Therefore, the initial concentration of HD-IA in the reaction solution at this time was 1.65 mM. Subsequently, the reaction solution was allowed to stand at 37 ° C. for 3 minutes, 5 minutes, 10 minutes, 30 minutes and 60 minutes from the start of the reaction to cause desulfurization reaction of HD-IA with rhI2S. Next, the reaction solution was heated at 100 ° C. for 5 minutes to deactivate the enzyme and stop the reaction.
〔酵素反応２〕
基質原液を，ＨＤ−Ｉ−Ａの濃度が０．１５，０．３０，０．４５，０．６０，０．７５及び１．５ｍＭとなるように，希釈用緩衝液で希釈して基質溶液とした。各濃度の基質溶液を５０μＬずつ試験管にとり３７℃に加温し，これに上記の３７℃に加温したｒｈＩ２Ｓ溶液を２５μＬずつ添加して混和して反応溶液とした。従って，このときの反応溶液中のＨＤ−Ｉ−Ａの初濃度は，各々，０．１０，０．２０，０．３０，０．４０，０．５０及び１．０ｍＭであった。次いで，反応溶液を３７℃で５分間静置して，ｒｈＩ２ＳによるＨＤ−Ｉ−Ａの脱硫化反応をさせた。次いで，反応溶液を１００℃で５分間加熱して酵素を失活させて反応を停止させた。[Enzyme reaction 2]
Substrate solution is diluted with dilution buffer so that the concentration of HD-IA is 0.15, 0.30, 0.45, 0.60, 0.75 and 1.5 mM. It was. 50 μL of each concentration of substrate solution was placed in a test tube and heated to 37 ° C., and 25 μL of the above rhI2S solution heated to 37 ° C. was added and mixed to prepare a reaction solution. Therefore, the initial concentrations of HD-IA in the reaction solution at this time were 0.10, 0.20, 0.30, 0.40, 0.50, and 1.0 mM, respectively. Next, the reaction solution was allowed to stand at 37 ° C. for 5 minutes to cause desulfurization of HD-IA using rhI2S. Next, the reaction solution was heated at 100 ° C. for 5 minutes to deactivate the enzyme and stop the reaction.
〔酵素反応速度の測定〕
（１）装置
島津ＨＰＬＣシステムＬＣ−２０Ａ（島津製作所）に，疎水性カラムクロマトグラフィー用カラム（ＣＨＥＭＣＯＳＯＲＢＴＭ ３００−７Ｃ４，４．６ｍｍ Ｉ．Ｄ．×５０ｍｍ，充填剤：ブチル化シリカゲル，ケムコ製）と，その下流に順相／親水性相互作用クロマトグラフィー用カラム（ＴＳＫｇｅｌ ＮＨ２−１００，４．６ｍｍ Ｉ．Ｄ．×１５０ｍｍ，充填剤：アミノ基を結合させたシリカゲル，東ソー製）とをセットした。カラムオーブンでカラムを５０℃に加熱するとともに，順相／親水性相互作用クロマトグラフィー用カラムの出口からの流路に吸光光度計を設置し，カラム通過後の溶液の，波長２３２ｎｍにおける吸光度を連続的に測定できるようにした。[Measurement of enzyme reaction rate]
(1) Apparatus Shimadzu HPLC system LC-20A (Shimadzu Corporation) is connected to a column for hydrophobic column chromatography (CHEMCOSORB ™ 300-7C4, 4.6 mm ID × 50 mm, packing material: butylated silica gel, manufactured by Chemco). If, downstream the normal phase / hydrophilic interaction chromatography column: set (TSKgel NH 2 -100,4.6mm I.D. × 150mm , fillers silica gel, manufactured by Tosoh bound with amino group) and did. While heating the column to 50 ° C in a column oven, an absorptiometer is installed in the flow path from the outlet of the column for normal phase / hydrophilic interaction chromatography, and the absorbance of the solution after passing through the column is continuously measured at a wavelength of 232 nm. So that it can be measured automatically.
（２）操作手順
移動相を，０．５ｍＬ／分の流速で上記カラムに流し，カラムを平衡化させた。次いで，同流速で，１０μＬの酵素反応終了後の反応溶液及び標準溶液を各々カラムに負荷し，更に，同流速で，移動相を５０分間流した。このとき，カラム通過後の溶液の波長２３２ｎｍにおける吸光度を連続的に測定してチャート上に検出するようにし，ＨＤ−Ｉ−ＡがｒｈＩ２Ｓにより脱硫化されて生じたＨＤ−ＩＩ−Ａに由来するピークの，チャート上の面積を求めた。標準溶液を分析したときのＨＤ−ＩＩ−Ａに由来するピーク面積との比較から，酵素反応により生じた各反応溶液中のＨＤ−ＩＩ−Ａの濃度（単位：ｍＭ）を算出した。(2) Operating procedure The mobile phase was passed through the column at a flow rate of 0.5 mL / min to equilibrate the column. Next, 10 μL of the reaction solution after completion of the enzyme reaction and the standard solution were loaded onto the column at the same flow rate, and the mobile phase was allowed to flow at the same flow rate for 50 minutes. At this time, the absorbance at a wavelength of 232 nm of the solution after passing through the column is continuously measured and detected on the chart, and HD-IA is derived from HD-II-A produced by desulfurization with rhI2S. The area of the peak on the chart was obtained. From the comparison with the peak area derived from HD-II-A when the standard solution was analyzed, the concentration (unit: mM) of HD-II-A in each reaction solution produced by the enzyme reaction was calculated.
（３）Ｖｍａｘ及びＫｍの決定
上記酵素反応２において，酵素反応時間が５分間であることから，酵素反応後の各反応溶液中のＨＤ−ＩＩ−Ａの濃度（単位：ｍＭ）を５で除した１分間当たりの当該濃度の変化値を，各反応溶液における反応速度Ｖ（単位：ｍＭ／分）として求めた。次いで，各反応溶液の基質の初濃度（反応開始前の基質濃度）をＳ０（単位：ｍＭ）とし，反応速度Ｖの逆数（１／Ｖ）を縦軸に，Ｓ０の逆数（１／Ｓ０）を横軸にプロット（Ｌｉｎｅｗｅａｖｅｒ−Ｂｕｒｋプロット）し，回帰分析を行った。こうして得られた直線のｙ軸との切片を１／Ｖｍａｘ（Ｖｍａｘは酵素反応の最大速度），ｘ軸との切片を−１／Ｋｍ（Ｋｍはミカエリス定数）として，ＶｍａｘとＫｍとを求めた。(3) Determination of Vmax and Km In the
〔解析結果〕
上記酵素反応１において，反応時間を６０分としたときの反応溶液を，カラムに通過させたときのチャートを図１に示した。図１において，ピークＡが基質のＨＤ−Ｉ−Ａに由来し，ピークＢがｒｈＩ２ＳによりＨＤ−Ｉ−Ａが脱硫化されて生じたＨＤ−ＩＩ−Ａに由来するものであった。反応時間（分）に対してピークＢの面積をプロットした結果，酵素反応１の反応条件において，反応開始から５〜１０分までの反応速度が，当該反応の初速度に近似することがわかった。〔Analysis result〕
In the enzyme reaction 1, a chart when the reaction solution when the reaction time is 60 minutes is passed through the column is shown in FIG. In FIG. 1, peak A was derived from the substrate HD-IA, and peak B was derived from HD-II-A produced by desulfurization of HD-IA by rhI2S. As a result of plotting the area of peak B against the reaction time (minutes), it was found that the reaction rate from the start of the reaction to 5 to 10 minutes approximated the initial rate of the reaction under the reaction conditions of enzyme reaction 1. .
そこで，上記酵素反応２において，反応時間を５分間に設定して，各反応溶液における反応の初速度を測定した。測定結果をＬｉｎｅｗｅａｖｅｒ−Ｂｕｒｋプロットしたものを図２に示した。回帰直線の相関係数は０．９９８３であり，最大速度（Ｖｍａｘ）は３４μＭ／分，ミカエリス定数（Ｋｍ）は１１７μＭであった。これらの結果は，基質として合成基質である４−メチルウンベリフェリル硫酸を使用することなく，ｒｈＩ２Ｓの天然基質と同一若しくは類似の構造を有する二糖を用いて，ｒｈＩ２Ｓの活性を測定できることを示す。 Therefore, in the
本発明によれば，Ｉ２Ｓの酵素活性を，生体内に存在するＩ２Ｓ本来の基質と構造が類似するか，若しくは構造が同一の基質を用いて測定することができるので，医薬品として製造したｒｈＩ２Ｓの品質をより適切に評価できる。 According to the present invention, the enzyme activity of I2S can be measured using a substrate having a structure similar to or identical to the original substrate of I2S present in the living body. The quality can be evaluated more appropriately.
Claims (4)
（ａ）イズロン酸−２−スルファターゼを，2位に硫酸基を有するウロン酸と，アミノ糖とが，１，４−グリコシド結合又は１，３−グリコシド結合により結合したものである二糖若しくはその塩を基質として，該基質と反応させるステップ，
（ｂ）上記ステップ（ａ）の反応により，該2位の硫酸基が脱硫酸化されることにより生じた生成物を，疎水性カラムクロマトグラフィー，続いて順相／親水性相互作用カラムクロマトグラフィーに付して，該基質から分離させるステップと，
（ｃ）上記ステップ（ｂ）のカラムクロマトグラフィーにより該基質から分離された該生成物を検出して，単位時間当たりに生成した該生成物の量を求めるステップとを，含んでなる方法であって，
該二糖が，下記の一般式（Ｉ）〜（ＩＶ）：
で示される群から選択される二糖若しくはその塩である，方法。 A method for measuring the enzymatic activity of iduronic acid-2-sulfatase, comprising the following steps:
(A) A disaccharide obtained by combining iduronic acid-2-sulfatase with a uronic acid having a sulfate group at the 2-position and an amino sugar by a 1,4-glycoside bond or a 1,3-glycoside bond, or Reacting a salt as a substrate with the substrate;
(B) The product produced by desulfation of the sulfate group at the 2-position by the reaction of step (a) is subjected to hydrophobic column chromatography , followed by normal phase / hydrophilic interaction column chromatography. Subjecting to separation from the substrate;
(C) detecting the products separated from said substrate by column chromatography in step (b), and determining the amount of said product produced per unit time, met method comprising And
The disaccharide is represented by the following general formulas (I) to (IV):
The method which is a disaccharide selected from the group shown by these, or its salt.
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