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大学・研究所にある論文を検索できる 「酵母オリゴ糖転移酵素の遺伝子点変異による、オリゴ糖転移活性と脂質結合型糖鎖の加水分解活性の脱共役 : オリゴ糖転移酵素研究に資する改良された酵素調製法とペプチド基質デザイン」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。

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酵母オリゴ糖転移酵素の遺伝子点変異による、オリゴ糖転移活性と脂質結合型糖鎖の加水分解活性の脱共役 : オリゴ糖転移酵素研究に資する改良された酵素調製法とペプチド基質デザイン

山﨑, 貴大 YAMASAKI, Takahiro ヤマサキ, タカヒロ 九州大学

2021.03.24

概要

オリゴ糖転移酵素(OST)は N 型糖鎖修飾における最初のステップを触媒する酵素で、糖タンパク質を作るためにアスパラギン残基にオリゴ糖鎖を転移する。OST 酵素はアクセプターであるアスパラギン残基が存在しない場合、糖鎖供与体の脂質結合型糖鎖(LLO)を加水分解して粗面小胞体内腔に遊離 N 型糖鎖(FNG)を恒常的に生成することが知られている。本研究では酵母の OST 酵素に含まれる触媒サブユニット STT3 タンパク質に高親和性のエピトープタグを付加することで、野生型 OST 酵素を共発現している酵母細胞から変異型 OST を選択して精製する方法を確立した。この方法によって、機能不全となった変異型 OST 酵素を調製することが可能になり、精製した OST を様々な生化学実験に使用できるようになった。変異型 STT3 サブユニットを含む OST 酵素について、in vitro で 2 つの活性を測定し、さらに同一変異を導入した酵母を用いて細胞内の N 型糖鎖修飾と FNG 量を測定した。その結果、変異を導入した酵母の表現型と細胞内の FNG 量の相関から、通常の培養条件では酵母の生育に FNG は必須ではないことが示唆された。さらに、アミノ酸保存モチーフであるDXD モチーフへの変異の導入はオリゴ糖転移活性に比べて加水分解活性を高めること、逆にDK モチーフへの変異の導入はオリゴ糖転移活性に比べて加水分解活性を低下させることがin vitro と in vivo の両方の条件で明らかになった。点突然変異の導入で OST 酵素の 2 つの活性の共役を解くことができるという知見は、将来的に酵素反応機構の解明に繋がる可能性がある。また、変異を含むOST 酵素が調製できたことで、15 残基の優れた性質をもつ基質ペプチドの発見とOST3サブユニットあるいは OST6 サブユニットのいずれかを含む 2 種類の OST 複合体の酵素特性の違いを明らかにできた。本研究で新規に見いだされた変異体、基質、方法は酵母や他の真核生物種における OST 酵素の分子基盤と生理学的役割の解明に有用である。

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