ALG11-CDG突变蛋白在原核细胞中的稳定表达  

Stable Expression of ALG11-CDG Mutant Proteins in Prokaryotic Cells

作　　者：王佩 李盛陶 王宁[1] 高晓冬[1] WANG Pei;LI Shengtao;WANG Ning;GAO Xiaodong(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2021年第2期65-72,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21778023,21807048);江苏省自然科学基金项目(BK20170174);中央高校自主科研计划项目(JUSRP11727);江苏高校品牌专业建设工程资助项目。

摘　　要：甘露糖基转移酶Alg11是N糖基化途径中的关键酶,催化第4和第5个甘露糖(Man)残基以α-1,2连键的形式转移到底物Man3-GlcNAc2-PP-Dol生成Man5-GlcNAc2-PP-Dol,人类ALG11基因致病突变会导致相关的先天性糖基化疾病ALG11-CDG(Congenital Disorders of Glycosylation)。目前,对ALG11-CDG的研究更侧重于其临床症状,而患者Alg11突变蛋白的性质还有待探索。作者以研究ALG11-CDG突变蛋白的重组表达及活性为目的,首先在人胚胎肾细胞HEK293和酿酒酵母中分别表达了Alg11野生型及突变体蛋白,发现其表达量存在显著差异,突变体蛋白不能稳定表达。最终在大肠杆菌中成功稳定表达了野生型及ALG11-CDG相关突变蛋白,利用液质联用定量分析了蛋白质的活性,结果显示,突变蛋白的活性存在不同程度的降低,其降低程度与ALG11-CDG疾病的严重程度存在一定的相关性。上述研究结果证明:1)ALG11-CDG相关突变蛋白的不稳定有可能是造成疾病的原因之一;2)体外定量测试大肠杆菌表达的ALG11-CDG相关突变蛋白的活性可以为相应患者的疾病严重程度提供参考。As a key enzymein the N-glycosylation pathway,mannosyltransferase Alg11 catalyzes the addition of 4th and 5th mannose residues to the substrate Man3-GlcNAc2-PP-Dol with theα-1,2 linkage,resulting Man5-GlcNAc2-PP-Dol.The mutations in human ALG11 gene will lead to the corresponding congenital disorders of glycosylation(CDG),namely ALG11-CDG.Currently,more attention was paid to the clinical symptoms in the study of ALG11-CDG,rather than the properties and functions of the mutant Alg11 proteins.This study aimed to investigate the expression of ALG11-CDG mutant proteins and test the activity.Firstly,the wild-type and mutant Alg11 proteins were expressed in human embryonic kidney cells(HEK293)and Saccharomyces cerevisiae.While in both expression systems,the mutant proteins showed great differences in expression level,indicating unstable expression.Alg11 and Alg11 mutated proteins were finally stably andsuccessfully expressed in E.coli,and the activity of mutated proteins was quantitatively analyzed by liquid and mass chromatography.The results showed that the activity of Alg11 and ALG11-CDG mutated proteins decreased to different degrees,which was correlated with the severity of ALG11-CDG disease.The results indicats that:1)the instability of mutant proteins may cause ALG11-CDG,2)the in vitro quantitative assay of the mutant proteins expressed in E.coli may provide an alternative method to analyze the severity of ALD11-CDG.

关 键 词：N-糖基化 糖基转移酶Alg11 ALG11-CDG 酶活 

分 类 号：Q786[生物学—分子生物学]