OGA糖苷酶酶活中心的鉴定  

Location of Glycosidase Catalytic Site of O-GlcNAcase

作　　者：李国超[1] 徐䶮 田高飞[1] 李建华[1] 李延平[1] 冯涛[1] 李静 

机构地区：[1]南开大学药学院,天津市分子药物研究重点实验室,天津300071

出　　处：《南开大学学报（自然科学版）》2012年第2期7-11,16,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金(31000371);中央高校基本科研业务费专项基金(65011091)

摘　　要：N-乙酰葡萄糖胺(O-linkedN-acetylglucosamine,O-GlcNAc)修饰是一种重要的蛋白翻译后修饰,参与生物体中的多种细胞活动.O-GlcNAcase(OGA)是生物体内唯一水解蛋白O-GlcNAc修饰的糖苷酶,但该酶的糖苷酶活性中心一直存在争论.实验利用PCR的方法,获得3种不同缺失的人源OGA片段(OGA1:aa1～916;OGA2:aa1～677;OGA3:aa1～350),并将3种片段构建到质粒pET28a中;重组质粒转化大肠杆菌BL21后,进行诱导表达、纯化;以4-甲基伞形酮2-乙酰氨基-2-脱氧葡萄糖为底物测定不同OGA片段的糖苷酶酶学特征常数.结果发现OGA3仍保持糖苷酶的活性,因此OGA的糖苷酶活性中心位于N端1-350aa.O-linked N-acetylglucosamine （O-GlcNAc） is an important protein post-transla-tional modification, which is involved in many cellular processes. O-GlcNAcase （OGA） is the only enzyme responsible for removal of O-GlcNAc from protein. However, to date no experimental data clearly indicates the glycosidase catalytic site of O-GlcNAcase. Here, three coding sequences of human OGA fragments （OGA1 ： aal-916： OGA2 ： aal-677 ; OGA3. aal-350） were amplified by PCR and cloned into plasmid pET28a. After transformed three recombinant plas- mids into Escherichia coli BL21, His-tagged recombinant protein were expressed and purified. Using 4-MU-GlcNAc （ 4-methylumbelliferyl 2-acetamido-2-deoxy-fl-D-glucopyrano-side ） as substrate, the enzymatic characteristics of three OGA fragments were determined. This results indicated that OGA3 （1-350 aa） retained glycosidase catalytic activity and N-terminal aal-350 is the glycosidase domain.

关 键 词：O-GLCNACASE 缺失突变 酶学特性 

分 类 号：Q786[生物学—分子生物学]