JP2005046029A - Method for analyzing sugar chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing a sugar chain, by which the sugar chain can highly simply and sensitively be analyzed only by adding a liquid reaction reagent to a liquid specimen without needing the preliminary labeling or purification of the specimen with a highly specific sugar transferase, and in which a detection system can be downsized because of having a liquid-liquid reaction. <P>SOLUTION: This method for analyzing the sugar chain on the basis of the reactivity of the sugar transferase is based on the change in the reactivity of the sugar transferase before the action of the sugar transferase and after the action of the sugar transferase. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００７０】
次に、同反応条件により、１０〜１５０μＭのラクトース（Ｇａｌβ１−４Ｇｌｃ）溶液の測定を行った結果、いずれのラクトース濃度においてもＡ３４０の減少は全く見られなかった（図−６）。さらに、βガラクトシダーゼ（東洋紡社製）により、処理したラクトースの測定を行った。その結果、ラクトース濃度に応じて直線的にＡ３４０の減少が見られた（図−６）。本結果から、グリコシダーゼ消化により糖転移酵素の反応性が変化すること、その変化から、糖鎖構造の推測が可能となることが確認された。
【００７１】
実施例 ２ ルイス構造糖鎖混合液の糖転移酵素による糖鎖解析シミュレーション
次に、表２に記載した各々未知量１７種類のルイス関連構造糖鎖からなる混合溶液の種々の糖転移酵素による糖鎖解析についてのシミュレーションを行った。
【００７２】
【表２】

【００７３】
【表３】

【００７４】
（１） α １−３ フコシルトランスフェラーゼ ＶＩＩ を用いた解析
α１−３フコシルトランスフェラーゼＶＩＩ（以後、ＦＵＴ７と省略する場合もある）は、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒのみを基質として、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒを形成する酵素である（非特許文献２４参照）。従って、表２記載のルイス関連構造糖鎖混合溶液中のＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒだけを検出することができる（表３参照）。さらに、該糖鎖混合溶液をα１−３フコース構造及びα１−４フコース構造を加水分解するα１−３／４フコシダーゼ（以下、α３／４Ｆａｓｅと省略する場合もある）により処理すると、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−ＲだけがＦＵＴ７によって検出可能なＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ構造に変換される（表３参照）。従って、予めＦＵＴ７のＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線を作成しておくことにより、これらの定量が可能となる。すなわち、未処理の該糖鎖混合溶液、及びα３／４Ｆａｓｅにより処理された該糖鎖混合溶液を各々ＦＵＴ７を用いて測定し、未処理該糖鎖混合溶液の測定値がＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの量となり、α３／４Ｆａｓｅ処理該糖鎖混合溶液の測定値から未処理該糖鎖混合溶液の測定値を引いた値が、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒの量となる（表３参照）。
【００７５】
以上の様に、ＦＵＴ７とα３／４Ｆａｓｅを用いることにより該糖鎖混合溶液中のＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ及びＳｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒの検出及び定量が可能となる。
【００７６】
（２） β １−４ ガラクトシルトランスフェラーゼを用いた解析
β１−４ガラクトシルトランスフェラーゼ（以下、β１−４ＧａｌＴと省略する場合もある）はＧｌｃＮＡｃ−Ｒを基質として、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒを形成する酵素である。表２に記載の糖鎖混合溶液中β１−４ＧａｌＴにより検出されるのは、ＧｌｃＮＡｃ−Ｒのみである（表４参照）。さらに、該糖鎖混合溶液をβ１−４ガラクトース構造のみを特異的に加水分解するβ１−４ガラクトシダーゼ（以下、β４Ｇａｓｅと省略する場合もある）により処理すると、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒだけがβ１−４ＧａｌＴによって検出可能なＧｌｃＮＡｃ−Ｒ構造に変換される（表４参照）。また、さらに、該糖鎖混合溶液をβ４Ｇａｓｅ及びα３／４Ｆａｓｅにより処理すると、Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲとＧａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒのみが、β１−４ＧａｌＴによって検出可能なＧｌｃＮＡｃ−Ｒ構造に変換される（表−４参照）。さらに、該糖鎖混合溶液をβ１−３ガラクトース構造のみを特異的に加水分解するβ１−３ガラクトシダーゼ（以下、β３Ｇａｓｅと省略する場合もある）により処理すると、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒだけがβ１−４ＧａｌＴによって検出可能なＧｌｃＮＡｃ−Ｒ構造に変換される（表−４参照）。また、さらに、該糖鎖混合溶液をβ３Ｇａｓｅ及びα３／４Ｆａｓｅにより処理すると、Ｇａｌβ１−３ＧｌｃＮＡｃ−ＲとＧａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒのみが、β１−４ＧａｌＴによって検出可能なＧｌｃＮＡｃ−Ｒ構造に変換される（表−４参照）。
【００７７】
【表４】

【００７８】
従って、予めβ１−４ＧａｌＴのＧｌｃＮＡｃ−Ｒに対する検量線を作成しておくことにより、これらの定量が可能となる。すなわち、未処理の該糖鎖混合溶液、β４Ｇａｓｅにより処理された該糖鎖混合溶液、β４Ｇａｓｅとα３／４Ｆａｓｅにより処理された該糖鎖混合溶液、β３Ｇａｓｅにより処理された該糖鎖混合溶液、β３Ｇａｓｅとα３／４Ｆａｓｅにより処理された該糖鎖混合溶液を各々β１−４ＧａｌＴを用いて測定すると、未処理該糖鎖混合溶液の測定値がＧｌｃＮＡｃ−Ｒの量となり、β４Ｇａｓｅ処理該糖鎖混合溶液の測定値から未処理該糖鎖混合溶液の測定値を引いた値が、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの量となる（表−４参照）。さらに、β４Ｇａｓｅ及びα３／４Ｆａｓｅ処理該糖鎖混合溶液の測定値からβ４Ｇａｓｅ処理該糖鎖混合溶液の測定値を引いた値が、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒの量となる（表−４参照）。また、β３Ｇａｓｅ処理該糖鎖混合溶液の測定値から未処理該糖鎖混合溶液の測定値を引いた値が、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの量となり、β３Ｇａｓｅ及びα３／４Ｆａｓｅ処理該糖鎖混合溶液の測定値からβ３Ｇａｓｅ処理該糖鎖混合溶液の測定値を引いた値が、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの量となる（表−４参照）
【００７９】
以上の様に、β１−４ＧａｌＴとβ４Ｇａｓｅ、β３Ｇａｓｅ及びα３／４Ｆａｓｅを用いることにより該糖鎖混合溶液中のＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＧａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの検出及び定量が可能となる。
【００８０】
（３） α １−３ フコシルトランスフェラーゼ ＩＸ を用いた解析
α１−３フコシルトランスフェラーゼＩＸ（以下、ＦＵＴ９と省略する場合もある）はＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ或いはＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒを基質として、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ或いはＦｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒを形成する酵素である（非特許文献２４参照）。
【００８１】
表−２に記載の糖鎖混合溶液中ＦＵＴ９によって検出されるのは、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒである（表−５参照）。さらに、該糖鎖混合溶液をβ４Ｇａｓｅにより処理すると、Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲはＦＵＴ９で検出できないＧｌｃＮＡｃ−Ｒに変換されるため、ＦＵＴ９によって検出されるのはＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒのみとなる（表−５参照）。また、該糖鎖混合溶液をβ４Ｇａｓｅ及びα３／４Ｆａｓｅにより処理すると、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−ＲがＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに変換され、糖鎖混合溶液中のＦＵＴ９により検出される糖鎖は、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−ＲとＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒとなる（表−５参照）。また、さらに、該糖鎖混合溶液をα１−３ガラクトースを特異的に加水分解するα１−３ガラクトシダーゼ（以下、α３Ｇａｓｅと省略する場合もある）により処理すると、Ｇａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲがＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに変換され、糖鎖混合溶液中のＦＵＴ９により検出される糖鎖は、Ｇａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒとなる（表−５参照）。また、さらに、該糖鎖混合溶液をα１−３Ｎ−アセチルガラクトサミンを特異的に加水分解するα１−３ Ｎ−アセチルがガラクトサミニダーゼ（以下、α３ＧＮａｓｅと省略する場合もある）により処理すると、ＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲがＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに変換され、糖鎖混合溶液中のＦＵＴ９により検出される糖鎖は、ＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒとなる（表−５参照）。
【００８２】
【表５】

【００８３】
従って、予めＦＵＴ９のＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線を作成しておくことにより、これらの定量が可能となる。すなわち、未処理の該糖鎖混合溶液、β４Ｇａｓｅにより処理された該糖鎖混合溶液、β４Ｇａｓｅ及びα３／４Ｆａｓｅにより処理された該糖鎖混合溶液、α３Ｇａｓｅにより処理された該糖鎖混合溶液、及びαＧＮａｓｅにより処理された該糖鎖混合溶液をＦＵＴ９を用いて測定し、それぞれの測定値から各成分の定量が可能となる。例えば、β４Ｇａｓｅ処理該糖鎖混合溶液の測定値から未処理該糖鎖混合溶液の測定値を差し引いた値をＧａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線を用いて定量することにより、該糖鎖混合溶液中のＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ量が定量される（表−５参照）。また、β４Ｇａｓｅ処理該糖鎖混合溶液の測定値をＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線用いて定量することにより該糖鎖混合溶液中のＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量が定量される（表−５参照）。また、β４Ｇａｓｅ及びα３／４Ｆａｓｅ処理該糖鎖混合溶液の測定値からβ４Ｇａｓｅ処理該糖鎖混合溶液の測定値を差し引いた値をＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線用いて定量することにより該糖鎖混合溶液中のＦｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ量が定量される（表−５参照）。また、α３Ｇａｓｅ処理該糖鎖混合溶液の測定値から未処理該糖鎖混合溶液の測定値を差し引いた値をＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線用いて定量することにより該糖鎖混合溶液中のＧａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量が定量される（表−５参照）。また、α３ＧＮａｓｅ処理該糖鎖混合溶液の測定値から未処該理糖鎖混合溶液の測定値を差し引いた値をＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線用いて定量することにより該糖鎖混合溶液中のＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量が定量される（表−５参照）。
【００８４】
以上の様に、ＦＵＴ９とβ４Ｇａｓｅ、α３Ｇａｓｅ、αＧＮａｓｅ及びα３／４Ｆａｓｅを用いることにより糖鎖混合溶液中のＦｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、ＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの検出及び定量が可能となる。
【００８５】
（４） α １−２ フコシルトランスフェラーゼを及びその他の糖転移酵素系を用いた解析
α１−２フコシルトランスフェラーゼ（以下、α１−２ＦｕｃＴと省略する場合もある）はＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ或いはＧａｌβ１−３ＧｌｃＮＡｃ−Ｒを基質として、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ或いはＦｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒを形成する酵素である（非特許文献２４参照）。
【００８６】
表−２に記載の糖鎖混合溶液中α１−２ＦｕｃＴによって検出されるのは、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ及びＧａｌβ１−３ＧｌｃＮＡｃ−Ｒである（表−６参照）。さらに、該糖鎖混合溶液をβ４Ｇａｓｅにより処理すると、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒはα１−２ＦｕｃＴで検出できないＧｌｃＮＡｃ−Ｒに変換されるため、α１−２ＦｕｃＴによって検出されるのはＧａｌβ１−３ＧｌｃＮＡｃ−Ｒのみとなる（表−６参照）。また、該糖鎖混合溶液をβ３Ｇａｓｅにより処理すると、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒはα１−２ＦｕｃＴで検出できないＧｌｃＮＡｃ−Ｒに変換されるため、α１−２ＦｕｃＴによって検出されるのはＧａｌβ１−４ＧｌｃＮＡｃ−Ｒのみとなる（表−６参照）。従って、予めα１−２ＦｕｃＴのＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ及びＧａｌβ１−３ＧｌｃＮＡｃ−Ｒに対する検量線を作成しておくことにより、これらの定量が可能となる。すなわち、未処理の該糖鎖混合溶液、β４Ｇａｓｅにより処理された該糖鎖混合溶液、及びβ３Ｇａｓｅにより処理された該糖鎖混合溶液をα１−２ＦｕｃＴを用いて測定し、それぞれの測定値から各成分の定量が可能となる。例えば、未処理該糖鎖混合溶液の測定値からβ４Ｇａｓｅ処理該糖鎖混合溶液の測定値を差し引いた値をＧａｌβ１−４ＧｌｃＮＡｃ−Ｒに対する検量線を用いて定量することにより、該糖鎖混合溶液中のＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ量が定量される（表−６参照）。また、未処理該糖鎖混合溶液の測定値からβ３Ｇａｓｅ処理該糖鎖混合溶液の測定値を差し引いた値をＧａｌβ１−３ＧｌｃＮＡｃ−Ｒに対する検量線を用いて定量することにより、該糖鎖混合溶液中のＧａｌβ１−３ＧｌｃＮＡｃ−Ｒ量が定量される（表−６参照）。
【００８７】
【表６】

【００８８】
また、該糖鎖混合溶液をα２−３シアル酸構造を特異的に加水分解するα２−３シアリダーゼ（以下、α３Ｓａｓｅと省略する場合もある）により処理すると、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−ＲがＧａｌβ１−３ＧｌｃＮＡｃ−Ｒ に、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲがＧａｌβ１−４ＧｌｃＮＡｃ−Ｒにそれぞれ変換され、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ及びＧａｌβ１−４ＧｌｃＮＡｃ−Ｒの構造に変化はなく、また、その他の成分もα１−２ＦｕｃＴに検出される構造に変換されないため、α３Ｓａｓｅにより処理した該糖鎖混合溶液をα１−２ＦｕｃＴを用いて測定して得られた測定値はＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＧａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和を示す（表−６参照）。また、該糖鎖混合溶液をα３Ｓａｓｅ及びα３／４Ｆａｓｅにより処理すると、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−ＲがＧａｌβ１−３ＧｌｃＮＡｃ−Ｒに、また、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲがＧａｌβ１−４ＧｌｃＮＡｃ−Ｒに変換され、さらに、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ及びＧａｌβ１−４ＧｌｃＮＡｃ−Ｒの構造に変化はなく、また、その他の成分もα１−２ＦｕｃＴに検出される構造に変換されないため、α３Ｓａｓｅ及びα３／４Ｆａｓｅにより処理した該糖鎖混合溶液をα１−２ＦｕｃＴを用いて測定して得られた測定値は、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＧａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和を示す（表−６参照）。そこで、α３Ｓａｓｅ及びα３／４Ｆａｓｅにより処理した該糖鎖混合溶液の測定値からα３Ｓａｓｅにより処理した該糖鎖混合溶液の測定値を差し引いた値は、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒの総和を示すことになる（表−６参照）。該糖鎖混合溶液中のＳｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（１）の方法により定量が可能であり、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、及びＧａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒの量は、上記実施例２−（２）の方法により測定が可能であるので、これらの方法によりＳｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、及びＧａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖混合溶液中のＳｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの量を定量することが可能となる。
【００８９】
以上の様に、α１−２ＦｕｃＴとβ３Ｇａｓｅ、β４Ｇａｓｅ、α３Ｓａｓｅ、及びα３／４Ｆａｓｅを用いることにより該糖鎖混合溶液中のＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの検出及び定量が可能となる。
【００９０】
（４） α １−３／４ フコシルトランスフェラーゼを及びその他の糖転移酵素系用いた解析
α１−３／４フコシルトランスフェラーゼ（以下、ＦＵＴ３と省略する場合もある）は、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−ＲのＧｌｃＮＡｃにα１−３結合でフコースを転移して、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒを生成するα１−３フコシルトランスフェラーゼ活性と、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−ＲのＧｌｃＮＡｃα１−４結合でフコースを転移して、Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒを生成するα１−４フコシルトランスフェラーゼ活性を有する酵素である（非特許文献２４参照）。従って、ＦＵＴ３のＧａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒに対する検量線を作成しておくことにより、上記実施例２−（１）〜（３）と同様に種々のグリコシダーゼとの組み合わせにより糖鎖の解析が可能となる。
【００９１】
例えば、表−２記載のルイス関連構造糖鎖混合溶液をα１−２フコース構造を特異的に加水分解するα１−２フコシダーゼ（以下、α２Ｆａｓｅと省略する場合もある）により処理した後、加熱処理などにより一旦α２Ｆａｓｅを失活させた後、β３Ｇａｓｅで処理したものをＦＵＴ３を用いて測定した測定値は、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和となる（表−７参照）。ここで、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（１）の方法により定量可能であり、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量、及びＧａｌβ１−３ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（２）の方法により定量可能であり、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（３）の方法により定量可能であることから、本測定値からＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖混合溶液中のＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの定量が可能になる。
【００９２】
【表７】

【００９３】
また例えば、該糖鎖混合溶液をα３Ｓａｓｅにより処理した後、加熱処理などにより一旦α３Ｓａｓｅを失活させた後、β３Ｇａｓｅで処理したものをＦＵＴ３を用いて測定した測定値は、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和となる（表−７参照）。ここで、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（１）の方法により定量可能であり、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（２）の方法により定量可能であり、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（３）の方法により定量可能であることから、本測定値からＳｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＦｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖混合溶液中のＦｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの定量が可能になる。
【００９４】
また例えば、該糖鎖混合溶液をα３Ｇａｓｅにより処理したものをＦＵＴ３を用いて測定した測定値は、Ｇａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＧａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和となる（表−７参照）。ここで、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの量は本実施例で上述された方法により定量可能であり、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（１）の方法により定量可能であり、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（２）の方法により定量可能であり、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（３）の方法により定量可能であることから、本測定値からＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＧａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖庫運号溶液中のＧａｌα１−３（Ｆｕｃα１−２）Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの定量が可能になる。
【００９５】
また例えば、該糖鎖混合溶液をα３ＧＮａｓｅにより処理したものをＦＵＴ３を用いて測定した測定値は、ＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの総和となる（表−７参照）。ここで、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの量は本実施例で上述された方法により定量可能であり、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（１）の方法により定量可能であり、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（２）の方法により定量可能であり、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、ＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（３）の方法により定量可能であることから、本測定値からＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖混合溶液中のＧａｌＮＡｃα１−３（Ｆｕｃα１−２）Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの定量が可能になる。
【００９６】
また例えば、該糖鎖混合溶液をα３／４Ｆａｓｅとβ３Ｇａｓｅにより処理したものをＦＵＴ３を用いて測定した測定値は、Ｆｕｃα１−２Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの総和となる（表−７参照）。
【００９７】
ここで、Ｓｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒの量は本実施例で上述された方法により定量可能であり、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒの量は上記実施例２−（１）の方法により定量可能であり、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−ＲＲ量は上記実施例２−（２）の方法により定量可能であり、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（３）の方法により定量可能であり、Ｓｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ量は上記実施例２−（４）の方法により定量可能であることから、本測定値からＳｉａα２−３Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−３ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｓｉａα２−３Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ、Ｆｕｃα１−２Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ、及びＳｉａα２−３Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒ量を差し引くことにより、該糖鎖混合溶液中のＦｕｃα１−２Ｇａｌβ１−３（Ｆｕｃα１−４）ＧｌｃＮＡｃ−Ｒの定量が可能になる。
【００９８】
以上の様に、本実施例の（１）〜（４）の方法を組み合わせることにより、表−２記載のルイス関連構造糖鎖混合溶液の１７種類の糖鎖を全て検出、定量することが可能であることが確認された。このような結果を踏まえ、さらに、糖鎖の大きな構造を認識する糖転移酵素（例えば、α１−６フコシルトランスフェラーゼやβ１−４ Ｎ−アセチルグルコサミニルトランスフェラーゼＩＩＩ、或いはβ１−６ Ｎ−アセチルグルコサミニルトランスフェラーゼＶ等）を用いることで、更に詳細な情報を得ることができる。また、糖脂質に特異性の高い糖転移酵素と糖蛋白質に特異性の高い糖転移酵素を使い分けることによってもさらに詳細な解析が可能になる。また、さらに、エンド型グリコシダーゼを活用することによってもさらに詳細な解析が可能になると考えられる。
【００９９】
【発明の効果】
本発明の方法では、従来の方法とは異なり液体状の試料に液体状の反応試薬を添加するだけで、感度良く糖鎖の解析が可能となり、液体−液体の反応で有るため従来の方法とは異なり検出システムの微小化が可能となる。また、特異性の高い糖転移酵素を用いてため、試料を予め標識したり精製したりする必要はなく、非常に簡便に糖鎖の解析が可能となる。
【０１００】
解析システムの微小化が可能になるため、例えばμＴＡＳなどを用いることで他種類の糖転移酵素−グリコシダーゼのセットを一度に測定することができ、糖鎖の網羅的解析、すなわち、糖鎖シーケンサーの開発が可能になる。また、簡便に糖鎖変化を測定できるため種々の診断薬用途或いは健康診断装置としての利用も可能になる。また、微小化により、携帯性が向上することから携帯型健康診断装置或いは健康診断チップとして利用が可能である。
【図面の簡単な説明】
【図１】本図は、ＧＤＰ−フコースやＵＤＰ−ガラクトースなどのＮＤＰ−糖を糖供与体とする糖転移酵素を糖鎖検出用素子として用いた場合の糖鎖検出方法の一例を示すものである。
【図２】本図は、ＣＭＰ−ＳｉａなどのＣＭＰ−糖を供与体とする糖転移酵素を糖鎖検出用素子として用いた場合の糖鎖検出方法の一例を示すものである。
【図３】本図は、ＧＤＰ−フコースやＵＤＰ−ガラクトースなどのＮＤＰ−糖を糖供与体とする糖転移酵素を糖鎖検出用素子として用いた場合の糖鎖検出方法の一例を示すものであり、その中でも発光法による方法を示すものである。
【図４】本図は、β１−４ガラクトシルトランスフェラーゼを糖鎖検出素子として用いてグルコースを測定した結果を示すものである。
【図５】本図は、β１−４ガラクトシルトランスフェラーゼを糖鎖検出素子として用いてグルコースを測定した場合に、β１−４ガラクトシルトランスフェラーゼの基質となり得ない糖質が存在してもグルコースの検出には影響が無いことを示した図である。
【図６】本図は、ガラクトシダーゼを組み合わせてβ１−４ガラクトシルトランスフェラーゼを糖鎖検出素子として用いてラクトースを測定した結果を示すものであり、グリコシダーゼ消化により糖転移酵素の反応性が変化すること、その変化から、糖鎖構造の推測が可能となることを示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a rapid and simple detection method and analysis method.
[0002]
[Prior art]
Sugar chains are substances in which various monosaccharides are linked by glycosidic bonds, and recent research has shown that they play an important role in various biological phenomena such as development / differentiation, nervous system, immune system, and cancer metastasis. It has become clear. Therefore, application to various fields including pharmaceuticals is expected, and interest in this sugar chain is increasing more and more.
In order to put sugar chains into practical use in various fields, in addition to the development of molecular biological analysis methods and sugar chain synthesis techniques, structure analysis techniques for sugar chains are important. However, sugar chains are difficult to detect because they exhibit little color development, fluorescence, or UV absorption, and because they have a branched structure and a variety of binding modes between constituent monosaccharides, Analysis can be said to be more difficult than peptide analysis and nucleic acid analysis.
[0003]
Currently known sugar chain analysis methods are classified into two types: a method of analyzing sugar chains after labeling and a method of analyzing without labeling. In the two-dimensional map method, which is one of the analysis methods by labeling, sugar chains fluorescently labeled with a fluorescent substance 2-aminopyridine are analyzed by two types of HPLC columns with different modes, and the analytical values in each column are known. This is a method for estimating the sugar chain structure of a sample with reference to an analysis map (for example, see Non-Patent Documents 1 to 5). In addition, FACE method (Fluorophore Assisted Carbohydrate Electrophoresis), which is another method of analysis by labeling, is a method in which a sugar chain fluorescently labeled with a fluorescent substance having a charge is analyzed by electrophoresis, and the structure is similarly estimated. . These fluorescent labeling methods (see, for example, Non-Patent Documents 6 to 8) are often used because they have high sensitivity and can analyze a small amount of sample. However, glycoproteins or glycolipids from biological samples are used. In other words, pretreatments such as purification of the sugar chain, excision of the sugar chain from the purified sample, labeling reaction of the sugar chain, and crude purification after labeling are necessary, and therefore, the operation is complicated and time-consuming. In addition, since these methods are basically HPLC analysis or electrophoretic analysis, it is difficult to directly analyze an unpurified biological sample in which other types of sugar chains or fluorescent substances are mixed, and it can be said that it is a simple method. It was not a thing.
[0004]
There are two known methods for analyzing unlabeled sugar chains, one using an analytical instrument and the other using biofunctional molecules such as antibodies. The HPAE-PAD method (High Performance Anion Exchange Exchange Detection), which is one of the analysis methods using analytical instruments, utilizes the property that sugar chains exhibit anionic properties in alkaline solutions. In this method, analysis is performed by anion exchange chromatography using a mobile phase, and a sugar chain is detected by a pulsed amperometry detector (for example, see Non-Patent Document 9). This method is excellent in that it can be analyzed without labeling sugar chains, but requires a special device, is inferior to the fluorescence labeling method in sensitivity, and it is difficult to prepare an unlabeled standard substance However, there was a problem such as low information due to low penetration rate. Further, as in the fluorescence labeling method, direct analysis of an unpurified biological sample is difficult and cannot be said to be a simple method. As other instrumental analysis methods, the development of NMR methods (for example, see Non-Patent Document 10) and mass spectrometry methods (for example, Non-Patent Documents 11 to 13) are progressing. None of the devices have been completed, and again, it has the disadvantage that direct analysis of unpurified biological samples is difficult.
[0005]
Unlike methods using instrumental analysis as described above, methods using biofunctional molecules are considered to be capable of direct analysis of unpurified biological samples because they utilize the high specificity and molecular recognition ability of living organisms. As these biofunctional molecules, lectins, which are proteins that specifically recognize sugar chains and bind / crosslink, and anti-sugar chain antibodies are often used for detection and analysis of sugar chains. However, although these antibodies and lectins specifically recognize and bind to sugar chains, they only bind and do not emit any signal that can detect the binding. Even if a molecular solution was added and reacted, it was impossible to detect in the solution whether or not it was bound. Therefore, in order to detect the binding, the reaction must be performed using a target biological sample or an immobilized biological molecule such as an antibody or a lectin. After the molecule is bound to the sugar chain, the biomolecule bound by another technique such as a secondary antibody must be detected, which is very complicated.
[0006]
As described above, it has been a reality that a sugar chain can be easily and rapidly analyzed with high sensitivity, and a technique for directly analyzing a sugar chain of an unpurified biological sample has not been developed.
[0007]
[Non-Patent Document 1]
“Biochemical and Biophysical Research Communications”, 85, p257, 1978. Biochemical and Biophysical Research Communications.
[0008]
[Non-Patent Document 2]
"Analytical Biochemistry", 163, p489, 1987
[0009]
[Non-Patent Document 3]
"Analytical Biochemistry", Vol.171, p73, 1988
[0010]
[Non-Patent Document 4]
"Analytical Biochemistry", 226, p130, 1995
[0011]
[Non-Patent Document 5]
"Biochemical Experimental Method 23, Glycoprotein Glycosid Research Method", Japan Society of Science Publishing Center, 1989
[0012]
[Non-Patent Document 6]
“Journal of Chromatography”, 646, p227, 1993
[0013]
[Non-Patent Document 7]
“Journal of Chromatography”, 705, p89, 1995.
[0014]
[Non-Patent Document 8]
“Journal of Chromatography”, 720, p295, 1996
[0015]
[Non-patent document 9]
"Method in Enzymology", 179, p65, 1989
[0016]
[Non-Patent Document 10]
"Advanceds in carbhydrate chemistry and biochemistry", 41, p209, 1983
[0017]
[Non-Patent Document 11]
"Advanceds in carbhydrate chemistry and biochemistry", 45, p19, 1987
[0018]
[Non-Patent Document 12]
“Journal of Chromatography B”, 734, p169, 1999
[0019]
[Non-Patent Document 13]
"Analytical Biochemistry", 207, p203, 1992
[0020]
[Non-Patent Document 14]
"Electrology E", 119, p476, 1999
[0021]
[Non-Patent Document 15]
"Proceedings of the Micro-TAS '98 Workshop, Banff, Canada", p1, 1998. Proceedings of the Micro-TAS '98 Workshop, Banff, Canada.
[0022]
[Non-Patent Document 16]
"Analytical chemistry, 71, p566, 1999
[0023]
[Non-Patent Document 17]
"Analytical chemistry, 70, p684, 1998
[0024]
[Non-Patent Document 18]
"Analytical chemistry, 70, p 5172, 1998
[0025]
[Non-Patent Document 19]
"Analytical chemistry, 70, p3790, 1998
[0026]
[Non-Patent Document 20]
"Analytical chemistry, 71, p4669, 1999
[0027]
[Non-patent document 21]
"Handbook of Glycosyltransferase and Related Genes", Springer, 2001
[0028]
[Non-Patent Document 22]
"Analytical Biochemistry", 36, p43, 1970
[0029]
[Non-Patent Document 23]
“Current Opinion in Drug Discovery & Development”, 3, p 756, 2000
[0030]
[Non-patent Document 24]
“Protein Nucleic Acid Enzyme December 1998 Special Issue Glycobiology From Glycan Information Transmission to Reception Mechanism”, Kyoritsu Shuppan Co., Ltd., p130, 1998
[0031]
[Means for Solving the Problems]
It is clear that glycan analysis using biofunctional molecules is useful in that analysis of an unpurified biological sample is possible and pretreatment such as labeling is unnecessary. Biofunctional molecules that can be used for detection of sugar chains, that is, biofunctional molecules that act on sugar chains include the above-mentioned lectins, anti-glycan antibodies, sugar chain digestion enzymes (glycosidases), glycosyltransferases (glycosyltransferases) And sulfate group transferase. Among the biofunctional molecules of the present invention, the glycosyltransferase specifically recognizes a sugar chain and is a biofunctional molecule that emits a detectable signal by reaction (or binding) with a sugar chain. Pay attention.
[0032]
Glycosyltransferase is an enzyme that uses a sugar nucleotide as a sugar donor and links a sugar in a specific form at a specific position of a sugar chain that is a sugar acceptor. Acid or nucleoside monophosphate (hereinafter sometimes abbreviated as NDP / NMP) is produced. Since this NDP / NMP can be easily detected by various methods, it is possible to detect whether or not a sugar transfer reaction has occurred by detecting whether NDP / NMP has been generated. That is, a biological sample to be measured is added to a reaction solution containing a glycosyltransferase having a specific sugar chain structure as a substrate and a sugar nucleotide serving as a sugar donor of the glycosyltransferase, and the glycosyltransferase is added. NDP / NMP is measured after the reaction is performed under the conditions under which the enzyme can react or during the glycosyltransferase reaction, and if NDP / NMP is detected, the substrate of the glycosyltransferase in the biological sample It can be seen that the sugar chain structure is included. In addition, since the amount of NDP / NMP produced by the glycosyltransferase reaction is proportional to the concentration of the sugar chain structure in the biological sample, the sugar chain structure can be quantified simultaneously with the detection of the sugar chain structure.
[0033]
For example, β1-4 galactosyltransferase (hereinafter sometimes abbreviated as β1-4GalT), which is a kind of glycosyltransferase, is UDP-galactose (hereinafter abbreviated as UDP-Gal in the absence of α-lactalbumin). Β1 to N-acetylglucosamine (hereinafter also abbreviated as GlcNAc) of a sugar acceptor substrate GlcNAc-R (GlcNAc: N-acetylglucosamine, R: sugar, represents a sugar chain). -4 bond is an enzyme that transfers galactose (hereinafter sometimes abbreviated as Gal), and by the glycosyltransferase reaction, Galβ1-4GlcNAc-R (Gal: galactose, GlcNAc: N-acetylglucosamine, R: sugar, Sugar chain) and uridine 5′-diphosphate (hereinafter, sometimes referred to as UDP) There is generated. Therefore, after adding a biological sample to the reaction solution containing β1-4GalT and UDP-Gal and reacting at a suitable temperature for a certain period of time, or measuring UDP during the reaction, production of UDP is detected. When it is determined, GlcNAc-R (R represents a sugar or a sugar chain) is present in the biological sample. Further, GlcNAc-R can be quantified by measuring the production amount of UDP.
[0034]
In addition, more glycan structure information can be obtained by comparing the glycosyltransferase reactivity of a biological sample treated with glycosidase, an enzyme that specifically cleaves monosaccharides constituting sugar chains, and a biological sample that is not treated. . For example, when GlcNAc-R, Galβ1-4GlcNAc-R, and Galβ1-3GlcNAc-R are mixed in a biological sample, when the biological sample is measured using β1-4GalT, only GlcNAc-R is detected and quantified. The Next, after digesting the biological sample with β1-4 galactosidase that specifically cleaves β1-4 galactose and measuring the biological sample using β1-4GalT, the amount of Galβ1-4GlcNAc-R is β1-4GalT. Therefore, Galβ1-4GlcNAc-R can be detected and quantified. Similarly, by performing digestion with β1-3 galactosidase that specifically cleaves β1-3 galactose, the Galβ1-3GlcNAc-R structure can be detected and quantified. In addition, after treatment with fucosidase that specifically cleaves fucose (hereinafter sometimes abbreviated as Fuc), the reaction amount of β1-4GalT (UDP production amount) does not increase, so fucosylated GlcNAc-R Is confirmed not to exist. Thus, it is possible to obtain more sugar chain structure information by combining glycosidase digestion and glycosyltransferase.
[0035]
By combining several sets of glycosyltransferases and glycosidases like this, it is considered that more advanced sugar chain structure analysis is possible.
[0036]
As described above, it has been found that simple and rapid structural analysis and quantification of sugar chains are possible by using glycosyltransferase as a sugar chain sensor element and NDP / NMP as a reaction signal.
[0037]
That is, the present invention includes the following inventions.
[0038]
Item 1. A method for detecting a sugar chain based on the reactivity of glycosyltransferase.
Item 2. A method for analyzing a sugar chain based on a change in the reactivity of glycosyltransferase before a sugar-cleaving enzyme is acted on and after a sugar-cleaving enzyme is acted on.
Item 3. A method for analyzing a sugar chain by combining several methods according to claim 1 and / or several methods according to claim 2.
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the reactivity of glycosyltransferase is detected by detecting nucleoside diphosphate or nucleoside monophosphate generated by a glycosyltransferase reaction.
Item 5. The glycosyltransferase is at least one selected from the group consisting of galactosyltransferase, fucosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, mannosyltransferase, glucosyltransferase, and xylosyltransferase. Item 5. The method according to any one of Items 1 to 4.
Item 6. Item 2. The sugar-cleaving enzyme is at least one selected from the group consisting of galactosidase, fucosidase, sialidase (or neuraminidase), N-acetylglucosaminidase, N-acetylgalactosaminidase, mannosidase, glucosidase, xylosidase, and glucuronidase. 6. The method according to any one of 5.
Item 7. Item 7. The method according to any one of Items 4 to 6, wherein the detected nucleoside diphosphate or nucleoside monophosphate is at least one selected from the group consisting of UDP, GDP, and CMP.
Item 8. Convert the amount of nucleoside diphosphate or nucleoside monophosphate generated by the glycosyltransferase reaction into an amount of a substance that can be easily detected by at least one enzyme reaction, and measure the amount of the converted substance. Item 8. The method according to any one of Items 4 to 7, wherein the method comprises measuring the amount of nucleoside diphosphate or nucleoside monophosphate produced by a glycosyltransferase reaction.
Item 9. 9. The method according to claim 8, wherein the method for measuring the amount of the converted substance is at least one selected from the group consisting of absorbance change measurement, luminescence measurement, and electrical signal measurement. .
Item 10. Item 10. The method according to Item 9, wherein the UDP or GDP detection method comprises the following steps (A) to (C).
(A) A step of reacting UDP or GDP produced by a glycosyltransferase reaction with phosphoenolpyruvate in the presence of pyruvate kinase to produce UTP or GTP and pyruvate.
(B) A step of reacting pyruvic acid produced in the step (A) with NADH in the presence of lactate dehydrogenase to produce lactic acid and NAD.
(C) A step of measuring the amount of NADH reduced by the step (B).
Item 11. Item 10. The method according to Item 9, wherein the CMP detection method comprises the following steps (A) to (D).
(A) A step of reacting CMP and ATP produced by a glycosyltransferase reaction in the presence of myokinase to produce CDP and ADP.
(B) A step of reacting CDP and ADP produced in the step (A) with phosphoenolpyruvate in the presence of pyruvate kinase to produce CTP, ATP and pyruvate.
(C) A step of reacting pyruvic acid generated in the step (B) with NADH in the presence of lactate dehydrogenase to produce lactic acid and NAD.
(D) A step of measuring the amount of NADH reduced by the step (C).
Item 12. Item 10. The method according to Item 9, wherein the UDP or GDP detection method comprises the following steps (A) to (D).
(A) A step of reacting UDP or GDP produced by a glycosyltransferase reaction with phosphoenolpyruvate in the presence of pyruvate kinase to produce UTP or GTP and pyruvate.
(B) A step of inactivating the enzyme activity of pyruvate kinase after the step (A).
(C) A step of reacting UTP or GTP produced in the step (A) with ADP in the presence of nucleoside diphosphate kinase to produce UDP or GDP and ATP.
(D) A step of measuring the amount of ATP produced by the step (C) with luciferase.
Item 13. Item 10. The method according to Item 9, wherein the CMP detection method comprises the following steps (A) to (E).
(A) A step of reacting CMP and ATP produced by a glycosyltransferase reaction in the presence of myokinase to produce CDP and ADP.
(B) A step of reacting CDP and ADP produced in the step (A) with phosphoenolpyruvate in the presence of pyruvate kinase to produce CTP, ATP and pyruvate.
(C) A method of inactivating the enzyme activities of myokinase and pyruvate kinase after the step (B).
(D) A step of reacting CTP or ATP produced in the step (B) with ADP in the presence of nucleoside diphosphate kinase to produce CDP or ADP and ATP.
(E) A step of measuring the amount of ATP produced by the step (D) with luciferase.
Item 14. Item 14. A sugar chain analyzer using any one of the methods according to items 1 to 13.
Item 15. Item 14. A sugar chain sensor using the method according to any one of items 1 to 13.
Item 16. A sugar chain sensor chip using the method according to claim 1.
Item 17. A diagnostic agent using the method according to claim 1.
Item 18. A health diagnostic apparatus using the method according to claim 1.
Item 19. A health sensor using the method according to claim 1.
Item 20. A health diagnostic sensor chip using the method according to claim 1.
[0039]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a sugar chain analysis method utilizing the property that nucleoside diphosphate or nucleoside monophosphate (hereinafter sometimes abbreviated as NDP / NMP) is generated by the high substrate characteristics and reaction of glycosyltransferase, It is a sugar chain analysis method characterized by measuring NDP / NMP produced by reaction of glycosyltransferase reactivity with an unknown sample. Furthermore, the present invention is a sugar chain analysis method characterized by analyzing a difference in reactivity of various glycosyltransferases before and after performing various glycosidase digestions.
[0040]
Glycosyltransferase reaction of the present invention
In the present invention, a glycosyltransferase is a sugar nucleotide linked to a sugar chain serving as a sugar acceptor using a sugar nucleotide as a sugar donor, and a nascent sugar chain and a nucleoside diphosphate or nucleoside monophosphate (hereinafter abbreviated as NDP / NMP). Any form may be used as long as it has the property. The glycosyltransferase may be extracted from nature, may be produced by genetic recombination, and may be derived from any biological species. In addition, a single purified preparation is not necessarily required as long as the contaminating glycosyltransferase or the hybridizing glycosidase does not exist so as to affect the glycosyltransferase reaction. It is also possible to use a fused glycosyltransferase in which another protein or peptide is fused to a glycosyltransferase as long as it does not affect the glycosyltransferase reaction. Furthermore, it is also possible to use an immobilized glycosyltransferase in a form in which glycosyltransferase is bound to various supports. For example, when the sugar chain detection system of the present invention is miniaturized and analyzed as a biochip such as a micro total analysis system (hereinafter sometimes abbreviated as μTAS) (for example, see Non-Patent Documents 14 to 20), It may be a chip that supplies a glycosyltransferase solution to a glycosyltransferase reaction chamber, or a chip in which the glycosyltransferase in the glycosyltransferase chamber is immobilized. The glycosyltransferase used in the present invention preferably has a well-known substrate specificity. However, even a glycosyltransferase having an unclear substrate specificity can be confirmed by confirming the substrate specificity by a preliminary experiment. It can be used in the present invention.
[0041]
Examples of the glycosyltransferase used in the present invention include galactosyltransferase (hereinafter sometimes abbreviated as GalT), fucosyltransferase (hereinafter sometimes abbreviated as FucT), and sialyltransferase (hereinafter abbreviated as SiaT). ), N-acetylglucosaminyltransferase (hereinafter sometimes abbreviated as GlcNAcT), N-acetylgalactosaminyltransferase (hereinafter sometimes abbreviated as GalNAcT), mannosyltransferase (hereinafter abbreviated as ManT). ), Glucosyltransferase (hereinafter sometimes abbreviated as GlcT), xylosyltransferase (hereinafter also abbreviated as XylT) and the like can be used (for example, see Non-Patent Document 21). ), It can be used of any type as long in those resulting nucleoside diphosphate or monophosphate by transglycosylation reactions.
[0042]
For example, as GalT, β1-4 galactosyltransferase-I, β1-4 galactosyltransferase-II, β1-4 galactosyltransferase-III β1-4 galactosyltransferase-IV, β1-4 galactosyltransferase-V, β1-4 galactosyltransferase- VI β1-4 galactosyltransferase-VII, β1-3 galactosyltransferase-I, β1-3 galactosyltransferase-II, β1-3 galactosyltransferase-III, β1-3 galactosyltransferase-IV, β1-3 galactosyltransferase-V, α1- 3 galactosyltransferase and the like are exemplified (for example, see Non-Patent Document 21), but UDP-galacto (Hereinafter also abbreviated as UDP-Gal) as a sugar donor to transfer galactose and produce uridine 5′-diphosphate (hereinafter abbreviated as UDP) as a reaction product. Anything can be used.
[0043]
Further, for example, as FucT, α1-2 fucosyltransferase-I, α1-2 fucosyltransferase-II, α1-3 / 4 fucosyltransferase, α1-3 fucosyltransferase-IV, α1-3 fucosyltransferase-V, α1- 3 fucosyltransferase-VI, α1-3 fucosyltransferase-VII, α1-3 fucosyltransferase-IX, α1-6 fucosyltransferase and the like are exemplified (for example, see Non-Patent Document 21), but GDP-fucose (hereinafter referred to as GDP) -Fuc) may be used as a sugar donor to transfer fucose, and any guanosine 5'-diphosphate (hereinafter may be abbreviated as GDP) as a reaction product is used. Is possible .
[0044]
For example, as SiaT, α2-3 sialyltransferase-I (ST3Gal-I), α2-3 sialyltransferase-II (ST3Gal-II), α2-3 sialyltransferase-III (ST3Gal-III), α2-3 Sialyltransferase-IV (ST3Gal-IV), α2-3 sialyltransferase-V (ST3Gal-V), galactose α2-6 sialyltransferase-I (ST6Gal-I), N-acetylgalactosamine α2-6 sialyltransferase-I (ST6GalNAc -I), N-acetylgalactosamine α2-6 sialyltransferase-II (ST6GalNAc-II), N-acetylgalactosamine α2-6 sialyltransferase-I I (ST6GalNAc-III), N-acetylgalactosamine α2-6 sialyltransferase-IV (ST6GalNAc-IV), α2-8 sialyltransferase-I (ST8Sia-I), α2-8 sialyltransferase-II (ST8Sia-II), Examples include α2-8 sialyltransferase-III (ST8Sia-III), α2-8 sialyltransferase-IV (ST8Sia-IV), α2-8 sialyltransferase-V (ST8Sia-V) (for example, non-patent literature). 21), sialic acid is transferred using CMP-sialic acid (hereinafter sometimes abbreviated as CMP-Sia) as a sugar donor, and 5′-cytidylic acid (hereinafter sometimes abbreviated as CMP) as a reaction product. ) If it is possible to use in any one.
[0045]
For example, GlcNAcT includes β1-2 N-acetylglucosaminyltransferase-I (GnT-I), β1-2 N-acetylglucosaminyltransferase-II (GnT-II), β1-3 N-acetyl. Glucosaminyltransferase (i-GnT), fucose β1-3 N-acetylglucosaminyltransferase (Fringe), β1-4 N-acetylglucosaminyltransferase-III (GnT-III), β1-4 N-acetylgluco Saminyltransferase-IV (GnT-IV), β1-4 N-acetylglucosaminyltransferase-VI (GnT-VI), β1-6 N-acetylglucosaminyltransferase-V (GnT-V), I antigen formation β1-6 N- Cetylglucosaminyltransferase (I-GnT), core 2 forming β1-6 N-acetylglucosaminyltransferase-I, core 2 forming β1-6 N-acetylglucosaminyltransferase-II, core 2 forming β1-6 N -Acetylglucosaminyltransferase-III, α1-4 N-acetylglucosaminyltransferase and the like are exemplified (for example, see Non-Patent Document 21), but UDP-N-acetylglucosamine (hereinafter abbreviated as UDP-GlcNAc) In some cases, N-acetylglucosamine can be transferred using a sugar donor as a sugar donor, and UDP can be generated as a reaction product.
[0046]
Further, for example, as GalNAcT, β1-4 N-acetylgalactosaminyltransferase, α1-3 N-acetylgalactosaminyltransferase (blood group A antigen synthase), α1-3 N-acetylgalactosaminyltransferase ( (For example, refer to Non-Patent Document 21) and N-acetylgalactosamine using UDP-N-acetylgalactosamine (hereinafter sometimes abbreviated as UDP-GalNAc) as a sugar donor. Any substance can be used as long as it can transfer UDP and produce UDP as a reaction product.
[0047]
In addition, ManT can be used as long as it transfers mannose using GDP-mannose (hereinafter sometimes abbreviated as GDP-Man) as a sugar donor to produce GDP as a reaction product.
[0048]
Further, GlcT can be used as long as it transfers glucose using UDP-glucose (hereinafter sometimes abbreviated as UDP-Glc) as a sugar donor and generates UDP as a reaction product.
[0049]
Further, XylT can be used as long as it transfers UDP-xylose (hereinafter sometimes abbreviated as UDP-Xyl) as a sugar donor to transfer xylose and generates UDP as a reaction product.
[0050]
The glycosyltransferase reaction may be any conditions as long as it is a reaction condition for carrying out the glycosyltransferase reaction, but it is preferable to use conditions close to the optimum reaction conditions for the glycosyltransferase used. Further, it is more preferable if the reaction conditions are such that the glycosidase used in a pair can act. For example, the reaction temperature is preferably in the range of 20 to 45 ° C, more preferably 25 ° C to 42 ° C, and still more preferably 30 ° C to 40 ° C, and generally a reaction temperature around 37 ° C is often used. However, since the optimum reaction temperature varies depending on the type of glycosyltransferase, it is generally not possible to set a general temperature. Various glycosidases to be used depending on the glycosyltransferase used or in pairs. It is desirable to set appropriately according to the property. Also, for example, the reaction pH is preferably pH 4.0 to 9.0, more preferably optimum reaction pH 5.0 to 8.5, and still more preferably optimum reaction pH 5.5 to 8.0. However, it is desirable to set appropriately according to the kind of glycosyltransferase and various properties of the glycosidase used as a pair as well as the optimum reaction temperature.
[0051]
When a substance that inhibits the glycosyltransferase reaction is present in the biological sample, the glycosyltransferase reaction may be performed after removing the inhibitory substance by a simple treatment in advance. For example, when the inhibitor is an enzyme, it can be treated using heat treatment, solvent treatment, a small chromatographic carrier, or the like. For example, when the sugar chain detection system of the present invention is miniaturized and analyzed as a biochip such as μTAS (see, for example, Non-Patent Documents 14 to 20), the channel is heated up to the glycosyltransferase reaction chamber. A chamber may be provided, or a separation channel utilizing principles such as molecular sieving, ion exchange, hydrophobic separation, and various other types may be provided in the channel until the sample goes to the glycosyltransferase reaction chamber. . In addition, when the inhibitor is a low-molecular substance, it can be pretreated with a desalting column or the like, and when used as a μTAS, a molecular sieve is passed through the flow path until the sample goes to the glycosyltransferase reaction chamber, It is also possible to provide a separation channel utilizing principles such as ion exchange, hydrophobic separation, and various other types.
[0052]
Sugar nucleotide of the present invention
In the present invention, it is preferable to use a sugar nucleotide that is specific for the glycosyltransferase used. For example, UDP-Gal for GalT, GDP-Fuc for FucT, CMP-Sia for SiaT, UDP-GlcNAc for GlcNAcT, UDP-GalNAc for GalNAcT, and GDP-Man for ManT, UDP-Glc is preferably used in the case of GlcT, and UDP-Xyl is preferably used in the case of XylT. The sugar nucleotide concentration may be any conditions as long as it produces nucleoside diphosphate or nucleoside monophosphate by a glycosyltransferase reaction. Preferably, the sugar nucleotide concentration is ½ of the Km value for the sugar nucleotide of the glycosyltransferase used. More than twice, more preferably more than twice the Km value, even more preferably more than 5 times the Km value, and most preferably more than 10 times the Km value.
[0053]
Glycosidase used in the present invention
In the present invention, any glycosidase can be used as long as it hydrolyzes the glycosidic bond of the sugar chain. The glycosidase may be extracted from nature, may be produced by genetic recombination, and may be derived from any biological species. In addition, a single purified sample is not necessarily required as long as a contaminating enzyme that causes a reaction other than the target reaction does not exist so much as to significantly affect the glycosidase reaction or the glycosyltransferase reaction. It is also possible to use a fused glycosidase in which another protein or peptide is fused to a glycosidase as long as it does not affect the glycosidase reaction. Furthermore, it is also possible to use immobilized glycosidase in a form in which glycosidase is bound to various supports. In addition, for example, when analyzing the sugar chain detection system of the present invention as a microchip and analyzing it as a biochip such as μTAS (see, for example, Non-Patent Documents 14 to 21), a chip that supplies a glycosidase solution to a glycosidase reaction chamber Alternatively, the chip may be a chip in which the glycosidase in the glycosidase reaction chamber is immobilized.
[0054]
As the glycosidase, for example, galactosidase, fucosidase, sialidase (neuraminidase), N-acetylglucosaminidase, N-acetylgalactosaminidase, mannosidase, glucosidase, xylosidase, and the like are preferable. For example, it is preferable to separately use β1-3 galactosyltransferase, β1-4 galactosyltransferase, or β1-6 galactosyltransferase, rather than using β1-3, 4, 6 galactosyltransferase. Further, not only exoglycosidase but also endoglycosidase can be an effective means. In addition, it is preferable that the substrate specificity as a glycosidase is well known, but even a glycosidase whose substrate specificity is unclear can be used in the present invention by confirming the substrate specificity by preliminary experiments. Is possible. The glycosidase used is preferably one that can act under the reaction conditions of the glycosyltransferase used in a pair. For example, using a glycosidase having the enzymatic properties of optimum reaction pH 4.0 to 9.0, preferably optimum reaction pH 5.0 to 8.5, more preferably optimum reaction pH 5.5 to 8.0. Is desirable.
[0055]
When a substance that inhibits the sugar glycosidase reaction is present in the biological sample, the glycosidase reaction may be performed after removing the inhibitory substance in advance by a simple treatment. For example, when the inhibitor is an enzyme, it can be treated using heat treatment, solvent treatment, a small chromatographic carrier, or the like. For example, when the sugar chain detection system of the present invention is miniaturized and used as a biochip such as μTAS (for example, see Non-Patent Documents 14 to 20), a heating chamber is provided in the flow path until the sample goes to the glycosidase reaction chamber. Alternatively, a separation channel using a principle such as molecular sieving, ion exchange, hydrophobic separation, or other various types may be provided in the channel until the sample goes to the glycosidase reaction chamber. In addition, when the inhibitor is a low-molecular substance, it can be pretreated with a desalting column or the like. When it is used as a μTAS, a molecular sieve is used in the flow path until the sample goes to the glycosidase reaction chamber. It is also possible to provide a separation channel utilizing the principle of exchange, hydrophobic separation, and various other types.
[0056]
Detection of nucleoside diphosphate or nucleoside monophosphate in the present invention
The detection method of NDP / NMP in the present invention may be performed using any method known per se. Moreover, since GalT, GlcT, GlcNAcT, GalNAcT, and XylT use UDP-sugar as a sugar donor, the nucleoside diphosphate produced by these glycosyltransferase reactions (hereinafter sometimes abbreviated as NDP) is UDP. , FucT and ManT use GDP-sugar nucleoside as a sugar donor, so NDP generated by the reaction of these sugar transfer is GDP, and SiaT uses CMP-Sia as a sugar donor, and thus it is generated by SiaT reaction. Nucleoside monophosphate (hereinafter abbreviated as NMP) is CMP. That is, almost all glycosyltransferase reactions can be detected by detecting UDP, GDP, or CMP.
[0057]
For example, when detecting NDP such as UDP or GDP, NDP produced by glycosyltransferase reaction is present in the presence of phosphoenolpyruvate (hereinafter sometimes abbreviated as PEP) and pyruvate kinase (hereinafter abbreviated as PK). The resulting pyruvic acid is converted to pyruvic acid and nucleoside triphosphate (hereinafter sometimes abbreviated as NTP) by the action of lactate dehydrogenase (hereinafter abbreviated as LDH) in the presence of NADH. The amount of NDP can be measured as a decrease in NADH that can be easily detected by ultraviolet absorption (A340) measurement (FIG. 1 and FIG. 1). (Refer nonpatent literature 22). In addition, for example, CMP is detected by converting CMP generated by the SiaT reaction into ADP and CDP by the action of myokinase (hereinafter sometimes abbreviated as MK) in the presence of ATP, and then converting the generated CDP to PK in the presence of PEP. The amount of CMP can be easily detected by UV absorption (A340) measurement by performing a reaction for converting pyruvic acid and CTP into NAD and lactic acid by the action of LDH in the presence of NADH. It can be measured as a decrease in NADH (see FIG. 2 and Non-Patent Documents 22 to 23). In the case of this reaction system, ADP generated by the MK reaction is simultaneously converted by the PEP / PK system and NADH / LDH system present in the reaction solution, and the NADH is similarly reduced. It is done. If it is such a method, it will become possible to measure very simply by using the spectrophotometer which is a general analytical instrument.
[0058]
In addition, for example, NDP such as UDP or GDP can be detected by converting NDP produced by glycosyltransferase reaction into pyruvic acid and NTP by the action of PK in the presence of PEP, and then inactivating PK by various methods such as heating. After that, the NTP generated by the PK reaction is converted into NDP (UDP or GDP) and ATP by the action of nucleoside diphosphate kinase (hereinafter sometimes abbreviated as NDK) in the presence of ADP, and the generated ATP is converted into a luciferase detection system. Thus, the amount of NDP produced by the glycosyltransferase reaction can be detected as the amount of luminescence (see FIG. 3). CMP can also be detected as the amount of luminescence by adding an MK reaction system. With such a light emission method, detection with higher sensitivity becomes possible.
[0059]
Enzymes used for the enzyme conversion method as described above may be extracted from nature or produced by genetic recombination as long as the target enzyme reaction is performed, and may be derived from any species. It may be a thing. In addition, it is not always necessary to use a single purified preparation as long as a contaminating enzyme that causes a reaction other than the target reaction does not exist so as to significantly affect the NDP / NMP conversion reaction. It is also possible to use a fusion enzyme in which another protein or peptide is fused to these enzymes within a range that does not affect the NDP / NMP conversion reaction. Furthermore, it is also possible to use an immobilized enzyme in a form in which enzymes are bound to various supports. For example, when the sugar chain detection system of the present invention is miniaturized and used as a biochip such as μTAS (see, for example, Non-Patent Documents 14 to 20), the enzyme is supplied to the NDP / NMP detection reaction chamber. Even if it is a chip | tip, the chip | tip of the form by which the enzymes of a NDP / NMP detection reaction chamber are fix | immobilized in the reaction chamber may be sufficient.
[0060]
In addition to the methods described above, the NDP / NMP produced as it is or after some conversion by an enzymatic method or the like, the amount of NDP / NMP (ie, glycosyltransferase reaction amount, ie, sugar chain abundance) It is also possible to use a method of detecting as an electrical signal or a method of detecting as other optical signals such as SPR and laser beam.
[0061]
NDP / NMP may be measured simultaneously with the glycosyltransferase reaction or after the glycosyltransferase reaction. It is more preferable to simultaneously perform the glycosyltransferase reaction and the NDP / NMP detection reaction because of its simplicity. For example, when the sugar chain detection system of the present invention is miniaturized and used as a biochip such as μTAS (for example, non-patent References 14 to 21), the glycosyltransferase reaction chamber and the NDP / NMP detection reaction chamber may be performed in the same reaction chamber, and a reaction chamber in which glycosyltransferase or converting enzymes are immobilized is used. May be. In addition to the glycosyltransferase reaction chamber and the NDP / NMP detection reaction chamber, a signal detection chamber (flow path) may be provided.
[0062]
In addition, for example, when an inhibitor of NDP / NMP detection reaction is present in a biological sample, after the glycosyltransferase reaction, the reaction solution is previously removed by simple treatment, and then NDP / NMP detection may be performed. Is possible. For example, when the inhibitor is an enzyme, it can be treated using heat treatment, solvent treatment, a small chromatographic carrier, or the like. In addition, for example, when the sugar chain detection system of the present invention is miniaturized and used as a biochip such as μTAS (see, for example, Non-Patent Documents 14 to 21), the flow path until the sample goes to the NDP / NMP detection reaction chamber A heating chamber may be provided, or a separation channel that utilizes principles such as molecular sieving, ion exchange, hydrophobic separation, and various other types may be provided in the channel until the sample goes to the NDP / NMP detection reaction chamber. It is. In addition, when the inhibitor is a low-molecular substance, it can be pretreated with a desalting column or the like. For example, when it is used as μTAS (see, for example, Non-Patent Documents 14 to 21), the sample is NDP. It is also possible to provide a separation channel using the principle such as molecular sieving, ion exchange, hydrophobic separation, and various other types in the channel leading to the / NMP detection reaction chamber.
[0063]
Use form of sugar chain detection system of the present invention
Any use form of the present invention may be used. For example, a glycosidase-treated or untreated sample is added to a test tube containing a glycosyltransferase reaction reagent and an NDP / NMP detection reaction reagent, and after the reaction, the reaction solution is transferred to a cuvette and NDP / NMP is measured. Can also be implemented. It can also be carried out by performing the reaction in a cuvette and measuring NDP / NMP simultaneously with the reaction. In addition, the sample is reacted in a test tube containing a glycosyltransferase reaction reagent and then subjected to some treatment, or left untreated and added to a test tube containing an NDP / NMP detection reaction reagent. It can also be carried out by transferring the liquid to a cuvette and measuring NDP / NMP, or by carrying out an NDP / NMP detection reaction in the cuvette and measuring NDP / NMP simultaneously with the reaction. These methods can also be carried out by continuously performing analysis using various automatic analyzers known per se.
[0064]
Further, since the sample is preferably as small as possible, it is preferable to further reduce the amount of reaction or detection. For example, the reaction is performed in a 96-well titer plate, or a measurement sample is added to the titer plate. It can also be implemented by measuring. In addition, since this method can measure not only a small amount of sample but also multiple samples at the same time, various glycosyltransferase-glycosidase reaction sets can be examined at one time. Becomes more convenient.
[0065]
Furthermore, by using the sugar chain detection system of the present invention as a microchip such as μTAS, the sample can be further micronized (see, for example, Non-Patent Documents 14 to 21). In the case of μTAS, a sample purification channel can also be introduced, and various samples can be handled. Furthermore, since a large amount of glycosyltransferase-glycosidase reaction set is incorporated into one chip, exhaustive analysis becomes possible, so that it can be used as a sugar chain sequencer. In addition, since the chip is miniaturized, the portability is increased, and it can be used as a portable health diagnostic device, and can also be used as a sugar chain sensor chip.
[0066]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, these are merely examples, and the scope of the present invention is not limited to the examples.
[0067]
Example 1 β 1-4 By galactosyltransferase and pyruvate kinase / lactate dehydrogenase reaction system UDP Detection and quantification of sugar chains using detection methods
50 mM Glycine / Glycine buffer pH 8.6 140 μM NADH (Sigma)-1.28 mM Phosphoenolurubic acid (Sigma)-5 mM Manganese chloride-50 mM KCl-370 μM UDP-Gal (Sigma)-0.2 % Α-lactalbumin (manufactured by Sigma)-7 U / ml pyruvate kinase (manufactured by Sigma)-10 U / ml lactate dehydrogenase (manufactured by Sigma)-12 mU / ml β1-4 galactosyltransferase (manufactured by Toyobo) 180 μl of a 10-150 μM glucose solution was added to the reaction solution consisting of the above, and the reaction solution with a total volume of 800 μl was reacted at 30 ° C., and the decrease in A340 in the reaction for 2 hours was measured (see Non-Patent Document 22). As a result, A340 decreased linearly according to the concentration of added glucose (FIG. 4). From this result, it was confirmed that glucose can be detected and quantified by this reaction system.
[0068]
Next, a 500 μM galactose solution, fucose solution, sialic acid solution, and N-acetylgalactosamine solution were measured under the same reaction conditions. As a result, no decrease in A340 was observed at any galactose concentration (Table 1). Furthermore, as a result of measuring a 10-150 μM glucose solution containing 100 μM galactose, fucose, sialic acid, and N-acetylgalactosamine under the same reaction conditions, a linear decrease in A340 was observed depending on the glucose concentration ( Fig. 5). From this result, it was confirmed that even glucose mixed solution can detect and quantify only glucose according to the characteristics of β1-4GalT.
[0069]
[Table 1]

[0070]
Next, as a result of measuring a 10-150 μM lactose (Galβ1-4Glc) solution under the same reaction conditions, no decrease in A340 was observed at any lactose concentration (FIG. 6). Furthermore, the treated lactose was measured with β-galactosidase (manufactured by Toyobo Co., Ltd.). As a result, A340 decreased linearly according to the lactose concentration (FIG. 6). From this result, it was confirmed that glycosylase digestion changed the reactivity of glycosyltransferase and that the sugar chain structure could be estimated from the change.
[0071]
Example 2 Simulation of sugar chain analysis by glycosyltransferase in Lewis structure sugar chain mixture
Next, a simulation was performed for sugar chain analysis using various glycosyltransferases in a mixed solution composed of 17 kinds of Lewis-related structural sugar chains each having an unknown amount shown in Table 2.
[0072]
[Table 2]

[0073]
[Table 3]

[0074]
(1) α 1-3 Fucosyltransferase VII Analysis using
α1-3 fucosyltransferase VII (hereinafter sometimes abbreviated as FUT7) is an enzyme that forms Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R using only Siaα2-3Galβ1-4GlcNAc-R as a substrate. (Refer nonpatent literature 24). Therefore, only Siaα2-3Galβ1-4GlcNAc-R in the Lewis related sugar chain mixed solution described in Table 2 can be detected (see Table 3). Further, when the sugar chain mixed solution is treated with α1-3 / 4 fucosidase that hydrolyzes α1-3 fucose structure and α1-4 fucose structure (hereinafter, may be abbreviated as α3 / 4Fase), Siaα2-3Galβ1- Only 4 (Fucα1-3) GlcNAc-R is converted to the Siaα2-3Galβ1-4GlcNAc-R structure detectable by FUT7 (see Table 3). Therefore, by preparing a calibration curve for FUT7 Siaα2-3Galβ1-4GlcNAc-R in advance, it is possible to quantify these. That is, the untreated sugar chain mixed solution and the sugar chain mixed solution treated with α3 / 4Fase were each measured using FUT7, and the measured value of the untreated sugar chain mixed solution was Siaα2-3Galβ1-4GlcNAc- The value obtained by subtracting the measured value of the untreated sugar chain mixed solution from the measured value of the α3 / 4Fase-treated sugar chain mixed solution is the amount of Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R. (See Table 3).
[0075]
As described above, by using FUT7 and α3 / 4Fase, Siaα2-3Galβ1-4GlcNAc-R and Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R in the sugar chain mixed solution can be detected and quantified. .
[0076]
(2) β 1-4 Analysis using galactosyltransferase
β1-4 galactosyltransferase (hereinafter sometimes abbreviated as β1-4GalT) is an enzyme that forms Galβ1-4GlcNAc-R using GlcNAc-R as a substrate. Only GlcNAc-R is detected by β1-4GalT in the sugar chain mixed solution described in Table 2 (see Table 4). Further, when the sugar chain mixed solution is treated with β1-4 galactosidase that specifically hydrolyzes only the β1-4 galactose structure (hereinafter sometimes abbreviated as β4Gase), only Galβ1-4GlcNAc-R becomes β1-4GalT. To a detectable GlcNAc-R structure (see Table 4). Further, when the sugar chain mixed solution is treated with β4Gase and α3 / 4Fase, only Galβ1-4GlcNAc-R and Galβ1-4 (Fucα1-3) GlcNAc-R can be detected by β1-4GalT. (See Table 4). Further, when the sugar chain mixed solution is treated with β1-3 galactosidase that specifically hydrolyzes only the β1-3 galactose structure (hereinafter sometimes abbreviated as β3Gase), only Galβ1-3GlcNAc-R becomes β1-4GalT. To a detectable GlcNAc-R structure (see Table 4). Further, when the sugar chain mixed solution is treated with β3Gase and α3 / 4Fase, only Galβ1-3GlcNAc-R and Galβ1-3 (Fucα1-4) GlcNAc-R can be detected by β1-4GalT. (See Table 4).
[0077]
[Table 4]

[0078]
Accordingly, by preparing a calibration curve for β1-4GalT with respect to GlcNAc-R in advance, it is possible to quantify these. That is, the untreated sugar chain mixed solution, the sugar chain mixed solution treated with β4Gase, the sugar chain mixed solution treated with β4Gase and α3 / 4Fase, the sugar chain mixed solution treated with β3Gase, β3Gase and When each of the sugar chain mixed solutions treated with α3 / 4Fase is measured using β1-4GalT, the measured value of the untreated sugar chain mixed solution becomes the amount of GlcNAc-R, and the measurement of the β4Gase treated sugar chain mixed solution is performed. The value obtained by subtracting the measured value of the untreated sugar chain mixed solution from the value is the amount of Galβ1-4GlcNAc-R (see Table 4). Further, the value obtained by subtracting the measured value of the β4Gase-treated sugar chain mixed solution from the measured value of the β4Gase- and α3 / 4Fase-treated sugar chain mixed solution is the amount of Galβ1-4 (Fucα1-3) GlcNAc-R (Table). -4). Also, the value obtained by subtracting the measured value of the untreated glycan mixed solution from the measured value of the β3Gase-treated glycan mixed solution is the amount of Galβ1-3GlcNAc-R, and the β3Gase and α3 / 4Fase-treated glycan mixed solution The value obtained by subtracting the measured value of the β3Gase-treated sugar chain mixed solution from the measured value is the amount of Galβ1-3 (Fucα1-4) GlcNAc-R (see Table 4).
[0079]
As described above, by using β1-4GalT and β4Gase, β3Gase and α3 / 4Fase, GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1-4 (Fucα1-3) GlcNAc-R, Galβ1 in the sugar chain mixed solution -3GlcNAc-R and Galβ1-3 (Fucα1-4) GlcNAc-R can be detected and quantified.
[0080]
(3) α 1-3 Fucosyltransferase IX Analysis using
α1-3 fucosyltransferase IX (hereinafter sometimes abbreviated as FUT9) is Galβ1-4GlcNAc-R or Fucα1-2Galβ1-4GlcNAc-R as a substrate, and Galβ1-4 (Fucα1-3) GlcNAc-R or Fucα1-2Galβ1. -4 (Fucα1-3) GlcNAc-R (see Non-Patent Document 24).
[0081]
It is Galβ1-4GlcNAc-R and Fucα1-2Galβ1-4GlcNAc-R that are detected by FUT9 in the sugar chain mixed solution described in Table-2 (see Table-5). Further, when the sugar chain mixed solution is treated with β4Gase, Galβ1-4GlcNAc-R is converted to GlcNAc-R that cannot be detected by FUT9, so that only Fucα1-2Galβ1-4GlcNAc-R is detected by FUT9 ( Table-5). Further, when the sugar chain mixed solution is treated with β4Gase and α3 / 4Fase, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R is converted to Fucα1-2Galβ1-4GlcNAc-R, and is converted by FUT9 in the sugar chain mixed solution. The sugar chains to be detected are Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R and Fucα1-2Galβ1-4GlcNAc-R (see Table-5). Further, when the sugar chain mixed solution is treated with α1-3 galactosidase that specifically hydrolyzes α1-3 galactose (hereinafter sometimes abbreviated as α3Gase), Galα1-3 (Fucα1-2) Galβ1- 4GlcNAc-R is converted to Fucα1-2Galβ1-4GlcNAc-R, and the sugar chains detected by FUT9 in the sugar chain mixed solution are Galα1-3 (Fucα1-2) Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, And Fucα1-2Galβ1-4GlcNAc-R (see Table-5). Further, when the sugar chain mixed solution is treated with galactosaminidase (hereinafter also abbreviated as α3GNase), α1-3 N-acetyl which specifically hydrolyzes α1-3N-acetylgalactosamine, GalNAcα1- 3 (Fucα1-2) Galβ1-4GlcNAc-R is converted to Fucα1-2Galβ1-4GlcNAc-R, and the sugar chain detected by FUT9 in the sugar chain mixed solution is GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAc- R, Galβ1-4GlcNAc-R, and Fucα1-2Galβ1-4GlcNAc-R (see Table-5).
[0082]
[Table 5]

[0083]
Accordingly, by preparing calibration curves for FUT9 Galβ1-4GlcNAc-R and Fucα1-2Galβ1-4GlcNAc-R in advance, these can be quantified. That is, the untreated sugar chain mixed solution, the sugar chain mixed solution treated with β4Gase, the sugar chain mixed solution treated with β4Gase and α3 / 4Fase, the sugar chain mixed solution treated with α3Gase, and αGNase The sugar chain mixed solution treated by the above is measured using FUT9, and each component can be quantified from each measured value. For example, by quantifying the value obtained by subtracting the measured value of the untreated glycan mixed solution from the measured value of the β4Gase-treated glycan mixed solution using a calibration curve for Galβ1-4GlcNAc-R, The amount of Galβ1-4GlcNAc-R is quantified (see Table-5). Further, the amount of Fucα1-2Galβ1-4GlcNAc-R in the sugar chain mixed solution is quantified by quantifying the measured value of the β4Gase-treated sugar chain mixed solution using a calibration curve for Fucα1-2Galβ1-4GlcNAc-R (Table See -5). In addition, by quantifying a value obtained by subtracting the measured value of the β4Gase-treated sugar chain mixed solution from the measured value of the β4Gase- and α3 / 4Fase-treated sugar chain mixed solution using a calibration curve for Fucα1-2Galβ1-4GlcNAc-R, The amount of Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R in the chain mixed solution is quantified (see Table-5). In addition, the value obtained by subtracting the measured value of the untreated sugar chain mixed solution from the measured value of the α3Gase-treated sugar chain mixed solution is quantified using a calibration curve for Fucα1-2Galβ1-4GlcNAc-R in the sugar chain mixed solution. The amount of Galα1-3 (Fucα1-2) Galβ1-4GlcNAc-R is quantified (see Table-5). In addition, by quantifying a value obtained by subtracting the measured value of the untreated glycan mixed solution from the measured value of the α3GNase-treated glycan mixed solution, using a calibration curve for Fucα1-2Galβ1-4GlcNAc-R, the glycan mixed solution The amount of GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAc-R in the medium is quantified (see Table-5).
[0084]
As described above, by using FUT9 and β4Gase, α3Gase, αGNase and α3 / 4Fase, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R, GalNAcα1-3 (Fucα1-2) Galβ1- in the sugar chain mixed solution 4GlcNAc-R, Galα1-3 (Fucα1-2) Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, and Fucα1-2Galβ1-4GlcNAc-R can be detected and quantified.
[0085]
(4) α 1-2 Analysis using fucosyltransferase and other glycosyltransferase systems
α1-2 fucosyltransferase (hereinafter, may be abbreviated as α1-2FucT) is obtained by using Galβ1-4GlcNAc-R or Galβ1-3GlcNAc-R as a substrate and Fucα1-2Galβ1-4GlcNAc-R or Fucα1-2Galβ1-3GlcNAc-R. It is an enzyme that forms (see Non-Patent Document 24).
[0086]
Galβ1-4GlcNAc-R and Galβ1-3GlcNAc-R are detected by α1-2FucT in the sugar chain mixed solution described in Table-2 (see Table-6). Further, when the sugar chain mixed solution is treated with β4Gase, Galβ1-4GlcNAc-R is converted to GlcNAc-R that cannot be detected by α1-2FucT, and therefore only Galβ1-3GlcNAc-R is detected by α1-2FucT. (See Table-6). In addition, when the sugar chain mixed solution is treated with β3Gase, Galβ1-3GlcNAc-R is converted to GlcNAc-R that cannot be detected by α1-2FucT, and therefore only Galβ1-4GlcNAc-R is detected by α1-2FucT. (See Table-6). Therefore, by preparing calibration curves for α1-2FucT for Galβ1-4GlcNAc-R and Galβ1-3GlcNAc-R in advance, it is possible to quantify these. That is, the untreated glycan mixed solution, the glycan mixed solution treated with β4Gase, and the glycan mixed solution treated with β3Gase were measured using α1-2FucT. Can be quantitatively determined. For example, by quantifying a value obtained by subtracting the measured value of the β4Gase-treated sugar chain mixed solution from the measured value of the untreated sugar chain mixed solution using a calibration curve for Galβ1-4GlcNAc-R, The amount of Galβ1-4GlcNAc-R is quantified (see Table-6). Further, by quantifying the value obtained by subtracting the measured value of the β3Gase-treated sugar chain mixed solution from the measured value of the untreated sugar chain mixed solution using a calibration curve for Galβ1-3GlcNAc-R, The amount of Galβ1-3GlcNAc-R is quantified (see Table-6).
[0087]
[Table 6]

[0088]
In addition, when the sugar chain mixed solution is treated with α2-3 sialidase (hereinafter also abbreviated as α3Sase) that specifically hydrolyzes the α2-3 sialic acid structure, Siaα2-3Galβ1-3GlcNAc-R becomes Galβ1- 3GlcNAc-R is converted to Siaα2-3Galβ1-4GlcNAc-R Siaα2-3Galβ1-4GlcNAc-R to Galβ1-4GlcNAc-R, respectively, and there is no change in the structure of Galβ1-3GlcNAc-R and Galβ1-4GlcNAc-R, Since other components are not converted to the structure detected by α1-2FucT, the measurement value obtained by measuring the sugar chain mixed solution treated with α3Sase using α1-2FucT is Siaα2-3Galβ1-3GlcNAc-R, Siaα2-3Gal 1-4GlcNAc-R, indicating the sum of Galβ1-3GlcNAc-R, and Galβ1-4GlcNAc-R (see Table 6). Further, when the sugar chain mixed solution is treated with α3Sase and α3 / 4Fase, Galβ1-3 (Fucα1-4) GlcNAc-R, Siaα2-3Galβ1-3 (Fucα1-4) GlcNAc-R, and Siaα2-3Galβ1-3GlcNAc- R is Galβ1-3GlcNAc-R, and Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, and Siaα2-3Galβ1-4GlcNAc-R are Galβ1-4GlcNAc-R. Furthermore, there is no change in the structures of Galβ1-3GlcNAc-R and Galβ1-4GlcNAc-R, and the other components are not converted into the structures detected by α1-2FucT, so that they are processed by α3Sase and α3 / 4Fase. The measured values obtained by measuring the treated sugar chain mixed solution using α1-2FucT were Galβ1-3 (Fucα1-4) GlcNAc-R, Siaα2-3Galβ1-3 (Fucα1-4) GlcNAc-R, Siaα2-3Galβ1-3GlcNAc-R, Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, -4GlcNAc-R is summed (see Table-6). Therefore, the value obtained by subtracting the measured value of the sugar chain mixed solution treated with α3Sase from the measured value of the sugar chain mixed solution treated with α3Sase and α3 / 4Fase is Galβ1-3 (Fucα1-4) GlcNAc-R, Galβ1. -4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-3 (Fucα1-4) GlcNAc-R, and Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R (Table-6) reference). The amount of Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R in the sugar chain mixed solution can be quantified by the method of Example 2- (1), and Galβ1-3 (Fucα1-4) GlcNAc-R , And Galβ1-4 (Fucα1-3) GlcNAc-R can be measured by the method of Example 2- (2), so that Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc can be measured by these methods. -R, Galβ1-3 (Fucα1-4) GlcNAc-R, and Galβ1-4 (Fucα1-3) GlcNAc-R are subtracted to give Siaα2-3Galβ1-3 (Fucα1-4) in the sugar chain mixed solution It becomes possible to quantify the amount of GlcNAc-R.
[0089]
As described above, by using α1-2FucT and β3Gase, β4Gase, α3Sase, and α3 / 4Fase, Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, and Siaα2-3Galβ1-3 (Fucα1 -4) GlcNAc-R can be detected and quantified.
[0090]
(4) α 1-3 / 4 Analysis using fucosyltransferase and other glycosyltransferase systems
α1-3 / 4 fucosyltransferase (hereinafter also abbreviated as FUT3) is α1-3 linked to GlcNAc of Galβ1-4GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and Siaα2-3Galβ1-4GlcNAc-R. Transfer fucose to produce Galβ1-4 (Fucα1-3) GlcNAc-R, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R, and Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R -3 fucosyltransferase activity and transfer of fucose at the GlcNAcα1-4 bond of Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R, and Siaα2-3Galβ1-3GlcNAc-R, and Galβ1-3 (Fu cα1-4) GlcNAc-R, Fucα1-2Galβ1-3 (Fucα1-4) GlcNAc-R, and Siaα2-3Galβ1-3 (Fucα1-4) GlcNAc-R are enzymes having α1-4 fucosyltransferase activity. (Refer nonpatent literature 24). Therefore, the amount of FUT3 to Galβ1-4GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R, and Siaα2-3Gcβ By creating a line, sugar chains can be analyzed by combining with various glycosidases in the same manner as in Examples 2- (1) to (3).
[0091]
For example, the Lewis-related structure sugar chain mixed solution described in Table 2 is treated with α1-2 fucosidase that specifically hydrolyzes the α1-2 fucose structure (hereinafter sometimes abbreviated as α2Fase), and then heat treatment, etc. After the inactivation of α2Fase by the above, the measured values obtained by using FUT3 after treatment with β3Gase were Siaα2-3Galβ1-3GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1- It is the sum of 3GlcNAc-R and Fucα1-2Galβ1-4GlcNAc-R (see Table-7). Here, the amount of Siaα2-3Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (1), and the amount of Galβ1-4GlcNAc-R and the amount of Galβ1-3GlcNAc-R are the same as those of Example 2- (2 ) And the amount of Fucα1-2Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (3) above. From this measured value, Siaα2-3Galβ1-4GlcNAc-R, Galβ1- By subtracting the amounts of 4GlcNAc-R, Galβ1-3GlcNAc-R, and Fucα1-2Galβ1-4GlcNAc-R, Siaα2-3Galβ1-3GlcNAc-R in the sugar chain mixed solution can be quantified.
[0092]
[Table 7]

[0093]
In addition, for example, after the sugar chain mixed solution is treated with α3Sase, α3Sase is once deactivated by heat treatment or the like and then treated with β3Gase, the measured value using FUT3 is Fucα1-2Galβ1-3GlcNAc- R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, and Fucα1-2Galβ1-4GlcNAc-R (see Table 7). Here, the amount of Siaα2-3Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (1), and the amount of Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (2). Since the amount of Fucα1-2Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (3) above, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, and Fucα1-2Galβ1 By subtracting the amount of -4GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R in the sugar chain mixed solution can be quantified.
[0094]
In addition, for example, measured values obtained by treating the sugar chain mixed solution with α3Gase using FUT3 are Galα1-3 (Fucα1-2) Galβ1-3GlcNAc-R, Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1 -3GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and Galα1-3 (Fucα1-2) Galβ1-4GlcNAc (See Table-7). Here, the amount of Siaα2-3Galβ1-3GlcNAc-R and Fucα1-2Galβ1-3GlcNAc-R can be quantified by the method described above in this example, and the amount of Siaα2-3Galβ1-4GlcNAc-R is the same as that of Example 2- ( The amount of Galβ1-4GlcNAc-R and Galβ1-3GlcNAc-R can be quantified by the method of Example 2- (2), and Fucα1-2Galβ1-4GlcNAc-R, Galα1-3 Since the amount of (Fucα1-2) Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (3), Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, By subtracting the amounts of alβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and Galα1-3 (Fucα1-2) Galβ1-4GlcNAc-R, Galα1 in the sugar chain transporting solution -3 (Fucα1-2) Galβ1-3GlcNAc-R can be quantified.
[0095]
In addition, for example, measured values obtained by treating the sugar chain mixed solution with α3GNase using FUT3 are GalNAcα1-3 (Fucα1-2) Galβ1-3GlcNAc-R, Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1 -3GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNA (See Table-7). Here, the amounts of Siaα2-3Galβ1-3GlcNAc-R and Fucα1-2Galβ1-3GlcNAc-R can be quantified by the method described above in this example, and the amount of Siaα2-3Galβ1-4GlcNAc-R is the same as in Example 2- ( The amount of Galβ1-4GlcNAc-R and Galβ1-3GlcNAc-R can be quantified by the method of Example 2- (2), and Fucα1-2Galβ1-4GlcNAc-R and GalNAcα1-3. Since the amount of (Fucα1-2) Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (3), Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R, Siaα2-3Galβ1-4GlcNAc By subtracting the amount of R, Galβ1-4GlcNAc-R, Galβ1-3GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAc-R, GalNAcα1 in the sugar chain mixed solution -3 (Fucα1-2) Galβ1-3GlcNAc-R can be quantified.
[0096]
Further, for example, measured values obtained by treating the sugar chain mixed solution with α3 / 4Fase and β3Gase using FUT3 are Fucα1-2Galβ1-3 (Fucα1-4) GlcNAc-R, Siaα2-3Galβ1-3GlcNAc-R , Fucα1-2Galβ1-3GlcNAc-R, Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1-4 (Fucα1-3) GlcNacR, -2Galβ1-4 (Fucα1-3) GlcNAc-R, Fucα1-2Galβ1-4GlcNAc-R, and Siaα2-3Galβ1-3 (Fucα1-4) GlcNAc-R (see Table 7).
[0097]
Here, the amount of Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R can be quantified by the method described above in this example, and Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2 The amount of -3Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (1), and the amount of Galβ1-4GlcNAc-R and Galβ1-4 (Fucα1-3) GlcNAc-RR is the same as that of Example 2- ( The amount of Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R and Fucα1-2Galβ1-4GlcNAc-R can be quantified by the method of Example 2- (3), and Siaα2 -3Galβ1-3 (Fucα1-4) GlcNAc-R amount is as above Since it can be quantified by the method of Example 2- (4), Siaα2-3Galβ1-3GlcNAc-R, Fucα1-2Galβ1-3GlcNAc-R, Siaα2-3Galβ1-4 (Fucα1-3) GlcNAc-R, Siaα2-3Galβ1-4GlcNAc-R, Galβ1-4GlcNAc-R, Galβ1-4 (Fucα1-3) GlcNAc-R, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R, Fucα1-2Galβ1-4GlcNAc2R By subtracting the amount of −3Galβ1-3 (Fucα1-4) GlcNAc-R, the quantification of Fucα1-2Galβ1-3 (Fucα1-4) GlcNAc-R in the sugar chain mixed solution becomes possible.
[0098]
As described above, by combining the methods (1) to (4) of this example, it is possible to detect and quantify all 17 types of sugar chains in the Lewis related structure sugar chain mixed solution described in Table-2. It was confirmed that. Based on these results, a glycosyltransferase that recognizes a large sugar chain structure (for example, α1-6 fucosyltransferase, β1-4 N-acetylglucosaminyltransferase III, or β1-6 N-acetylglucosa). More detailed information can be obtained by using minyltransferase V or the like. Further detailed analysis is also possible by using a glycosyltransferase having high specificity for glycolipid and a glycosyltransferase having high specificity for glycoprotein. Further, it is considered that further detailed analysis can be performed by utilizing endo-type glycosidase.
[0099]
【The invention's effect】
In the method of the present invention, unlike conventional methods, sugar chains can be analyzed with high sensitivity simply by adding a liquid reaction reagent to a liquid sample, and this is a liquid-liquid reaction. Unlike the above, the detection system can be miniaturized. In addition, since a highly specific glycosyltransferase is used, it is not necessary to label or purify the sample in advance, and sugar chains can be analyzed very simply.
[0100]
Since the analysis system can be miniaturized, a set of other types of glycosyltransferase-glycosidase can be measured at one time by using, for example, μTAS. Development becomes possible. In addition, since the sugar chain change can be easily measured, it can be used as various diagnostic agents or as a health diagnostic apparatus. Further, since the portability is improved by miniaturization, it can be used as a portable health diagnostic device or a health diagnostic chip.
[Brief description of the drawings]
FIG. 1 shows an example of a sugar chain detection method when a glycosyltransferase having an NDP-sugar such as GDP-fucose or UDP-galactose as a sugar donor is used as a sugar chain detection element. is there.
FIG. 2 shows an example of a sugar chain detection method when a glycosyltransferase having a CMP-sugar donor such as CMP-Sia is used as a sugar chain detection element.
FIG. 3 shows an example of a sugar chain detection method when a glycosyltransferase having an NDP-sugar such as GDP-fucose or UDP-galactose as a sugar donor is used as a sugar chain detection element. Among them, the method by the luminescence method is shown.
FIG. 4 shows the results of measuring glucose using β1-4 galactosyltransferase as a sugar chain detection element.
FIG. 5 shows that when glucose is measured using β1-4 galactosyltransferase as a sugar chain detection element, glucose can be detected even if a carbohydrate that cannot serve as a substrate for β1-4 galactosyltransferase is present. It is the figure which showed that there is no influence.
FIG. 6 shows the results of measuring lactose using β1-4 galactosyltransferase as a sugar chain detection element in combination with galactosidase, and the glycosidase digestion changes the reactivity of glycosyltransferase. It is a figure showing that the sugar chain structure can be estimated from the change.

Claims (20)

A method for detecting a sugar chain based on the reactivity of glycosyltransferase. A method for analyzing a sugar chain based on a change in the reactivity of glycosyltransferase before a sugar-cleaving enzyme is acted on and after a sugar-cleaving enzyme is acted on. A method for analyzing a sugar chain by combining several methods according to claim 1 and / or several methods according to claim 2. The method according to any one of claims 1 to 3, wherein the reactivity of glycosyltransferase is detected by detecting nucleoside diphosphate or nucleoside monophosphate produced by a glycosyltransferase reaction. The glycosyltransferase is at least one selected from the group consisting of galactosyltransferase, fucosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, mannosyltransferase, glucosyltransferase, and xylosyltransferase. The method according to any one of claims 1 to 4, wherein: The sugar-cleaving enzyme is at least one selected from the group consisting of galactosidase, fucosidase, sialidase (or neuraminidase), N-acetylglucosaminidase, N-acetylgalactosaminidase, mannosidase, glucosidase, xylosidase, and glucuronidase. The method in any one of -5. The method according to any one of claims 4 to 6, wherein the detected nucleoside diphosphate or nucleoside monophosphate is at least one selected from the group consisting of UDP, GDP and CMP. Convert the amount of nucleoside diphosphate or nucleoside monophosphate produced by the glycosyltransferase reaction into an easily detectable substance amount by at least one enzyme reaction, and measure the amount of the converted substance The method according to any one of claims 4 to 7, wherein the amount of nucleoside diphosphate or nucleoside monophosphate produced by the glycosyltransferase reaction is measured by the method. 9. The method according to claim 8, wherein the method for measuring the amount of the converted substance is at least one selected from the group consisting of absorbance change measurement, luminescence measurement, and electrical signal measurement. Method. The method according to claim 9, wherein the UDP or GDP detection method includes the following steps (A) to (C).
(A) A step of reacting UDP or GDP produced by a glycosyltransferase reaction with phosphoenolpyruvate in the presence of pyruvate kinase to produce UTP or GTP and pyruvate.
(B) A step of reacting pyruvic acid produced in the step (A) with NADH in the presence of lactate dehydrogenase to produce lactic acid and NAD.
(C) A step of measuring the amount of NADH reduced by the step (B). The method according to claim 9, wherein the CMP detection method includes the following steps (A) to (D).
(A) A step of reacting CMP and ATP produced by a glycosyltransferase reaction in the presence of myokinase to produce CDP and ADP.
(B) A step of reacting CDP and ADP produced in the step (A) with phosphoenolpyruvate in the presence of pyruvate kinase to produce CTP, ATP and pyruvate.
(C) A step of reacting pyruvic acid generated in the step (B) with NADH in the presence of lactate dehydrogenase to produce lactic acid and NAD.
(D) A step of measuring the amount of NADH reduced by the step (C). The method according to claim 9, wherein the UDP or GDP detection method includes the following steps (A) to (D).
(A) A step of reacting UDP or GDP produced by a glycosyltransferase reaction with phosphoenolpyruvate in the presence of pyruvate kinase to produce UTP or GTP and pyruvate.
(B) A step of inactivating the enzyme activity of pyruvate kinase after the step (A).
(C) A step of reacting UTP or GTP produced in the step (A) with ADP in the presence of nucleoside diphosphate kinase to produce UDP or GDP and ATP.
(D) A step of measuring the amount of ATP produced by the step (C) with luciferase. The method according to claim 9, wherein the CMP detection method includes the following steps (A) to (E).
(A) A step of producing CDP and ADP by reacting CMP and ATP produced by a glycosyltransferase reaction in the presence of myokinase.
(B) A step of reacting CDP and ADP produced in the step (A) with phosphoenolpyruvate in the presence of pyruvate kinase to produce CTP, ATP and pyruvate.
(C) A method of inactivating the enzymatic activities of myokinase and pyruvate kinase after the step (B).
(D) A step of reacting CTP or ATP produced in the step (B) with ADP in the presence of nucleoside diphosphate kinase to produce CDP or ADP and ATP.
(E) A step of measuring the amount of ATP produced by the step (D) with luciferase. A sugar chain analyzer using the method according to claim 1. A sugar chain sensor using the method according to claim 1. A sugar chain sensor chip using the method according to claim 1. A diagnostic agent using the method according to claim 1. A health diagnostic apparatus using the method according to claim 1. A health sensor using the method according to claim 1. A health diagnostic sensor chip using the method according to claim 1.

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