摘要
氧连接的β-N-乙酰葡萄糖胺修饰(O-GlcNAc修饰)是一种发生在蛋白质丝氨酸、苏氨酸羟基上的一种动态、可逆的翻译后修饰。O-GlcNAc修饰在细胞的许多关键过程中发挥重要作用,也是研究阿尔茨海默病和糖尿病等的重要途径,但是常用于O-GlcNAc蛋白检测的抗体特异性或者检测分子量范围仍存在问题且耗时(~36 h)过长,对后续研究造成困扰。在本研究中,我们设计构建了产气荚膜梭菌OGA(O-GlcNAcase)突变体(CpOGA^(D298N))与核心链霉亲和素Stv13融合表达质粒pET28a-CpOGA^(D298N)-Stv13。将质粒pET28a-CpOGA^(D298N)-Stv13转化到大肠杆菌BL21(DE3)中进行诱导表达,获得了融合蛋白质CpOGA^(D298N)-Stv13。通过CpOGA^(D298N)特异性结合O-GlcNAc的能力及生物素-亲和素特异性亲和作用,设计了Far-Western blot(Far-WB)用于检测蛋白质O-GlcNAc修饰,利用sOGT(short form O-GlcNAc transferase)、去O-GlcNAc修饰sOGT、细胞裂解液样本及人肝细胞核因子1A(hepatocyte fuclear factor 1A,HNF1A)对该方法进行验证。本研究成功构建了一种蛋白质O-GlcNAc修饰检测方法,耗时相对O-GlcNAc特异性抗体缩短至5~7 h,丰富了蛋白质O-GlcNAc修饰的检测方法,为其后续研究奠定了基础。
Oxygen-linkedβ-N-acetylglucosamine(O-GlcNAc)modification is a dynamic,reversible post-translational modification that occurs on the hydroxyl group of protein serine and threonine residues and plays important roles in many key cellular processes.Antibodies are commonly used in the detection of protein O-GlcNAcylation,but their specificity against O-GlcNAcylation or the molecular weight range of detected proteins remains to be improved.The Clostridium perfringens OGA mutant(CpOGA^(D298N))has been applied in the detection of O-GlcNAcylation in a far-Western blot(Far-WB),due to its advantage in the binding with O-GlcNAc.In this study,CpOGA^(D298N) was fused with a core streptavidin Stv13.Based on the specific binding property between biotin and streptavidin,we established a fast O-GlcNAc Far-WB assay method,which is verified using short OGT(sOGT),de-O-GlcNAc sOGT,cell lysates and HNF1A proteins.Taken together,our work provides a specific and time-saving Far-WB method(5-7 hours),which can be effectively used in the detection of protein O-GlcNAcylation.
作者
黎先感
杨明
鲁莹
李玲
马骞
李静
张连文
LI Xian-Gan;YANG Ming;LU Ying;LI Ling;MA Qian;LI Jing;ZHANG Lian-Wen(College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,Nankai University,Tianjin 300350,China;College of Life Sciences and Beijing Key Lab DNA Damage Response,Capital Normal University,Beijing 100048,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2024年第4期513-519,共7页
Chinese Journal of Biochemistry and Molecular Biology
