上海交通大学学报（医学版）

上海交通大学学报(医学版) ›› 2020, Vol. 40 ›› Issue (11): 1461-1467.doi: 10.3969/j.issn.1674-8115.2020.11.004

• 论著·基础研究 • 上一篇    下一篇

ALG3异常的先天性糖基化疾病相关突变蛋白活性的检测

罗冰洁，李盛陶，王 宁，高晓冬   

  1. 江南大学生物工程学院糖化学与生物技术教育部重点实验室, 无锡 214122
  • 出版日期:2020-11-28 发布日期:2021-01-13
  • 通讯作者: 高晓冬，电子信箱：xdgao@jiangnan.edu.cn。
  • 作者简介:罗冰洁（1994—），女，硕士生；电子信箱：513354158@qq.com。
  • 基金资助:
    国家自然科学基金（21778023，21807048）；江苏省自然科学基金（BK20170174）；中央高校自主科研计划（JUSRP11727）；江苏高校品牌专业建设工程资助项目。

Detection of activity of mutant protein in ALG3-CDG

LUO Bing-jie, LI Sheng-tao, WANG Ning, GAO Xiao-dong   

  1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2020-11-28 Published:2021-01-13
  • Supported by:
    National Natural Science Foundation of China (21778023, 21807048); Natural Science Foundation of Jiangsu Province (BK20170174); Fundamental Research Fund for the Central University (JUSRP11727); Top-notch Academic Program of Jiangsu Higher Education Institution.

PDF (PC)

305

摘要： 目的·研究α-1,3-甘露糖转移酶（α-1,3-mannosyltransferase，ALG3）基因异常导致的先天性糖基化疾病（ALG3-CDG）相关突变蛋白的活性检测方法，并检验其活性与疾病严重程度的关系。方法·收集文献报道的ALG3-CDG患者ALG3蛋白6个突变位点（I69T、W71R、G96R、M157K、R171Q和M209T），同源比对人和酿酒酵母ALG3蛋白的氨基酸序列，设计对应的酿酒酵母ALG3突变体（I70T、Y72R、G98R、L157K、R171Q和M221T），并在大肠埃希菌体系中表达。利用含有野生型ALG3或其突变蛋白的大肠埃希菌菌膜催化甘露糖基转移反应，采用液相色谱-质谱联用技术，以野生型蛋白催化活性为基准，计算突变蛋白的相对活性。比较不同生存期患者对应突变蛋白的活性。结果·ALG3-CDG患者6个突变对应的酿酒酵母ALG3突变蛋白均在大肠埃希菌体系表达，蛋白表达量与野生型基本一致。体外定量活性测试结果显示，与野生型蛋白相比，I70T、Y72R、G98R、L157K、R171Q、M221T突变蛋白的相对活性分别为2.8%、4.9%、4.5%、17.2%、4.8%、22.3%。生存期大于1年患者对应的突变蛋白活性显著高于小于1年的患者（P=0.002）。结论·建立了ALG3-CDG相关酿酒酵母保守突变蛋白的体外定量活性检测方法，为该病严重程度的预测提供了可能。

关键词: 先天性糖基化疾病, 甘露糖转移酶, 液相色谱-质谱联用技术, 原核表达

Abstract:

Objective · To investigate the activity detection method of α-1,3-mannosyltransferase (ALG3) mutant protein in congenital disorder of glycosylation (ALG3-CDG), and examine the relationship between the activity and the severity of disease. Methods · The mutation sites of the patients in the reports were I69T, W71R, G96R, M157K, R171Q and M209T, respectively. After homologous alignment of the amino acid sequences of Homo sapiens and Saccharomyces cerevisiae (S. cerevisiae) ALG3, six yeast mutant proteins (I70T, Y72R, G98R, L157K, R171Q and M221T) related to ALG3-CDG were designed. These mutants were expressed in Escherichia coli (E. coli). Using E. coli membrane containing wild-type ALG3 or its mutant proteins as catalysts, the relative activities of mutant proteins were calculated by liquid chromatography tandem mass spectrometry based on the catalytic activity of wild type protein. The activities of corresponding mutations in the patients with different survival time were compared. Results · Six ALG3-CDG related S. cerevisiae conserved ALG3 mutant proteins were expressed in E. coli in the comparable protein expression levels with wild type ALG3. The in vitro quantitative activity assay of enzymes showed that compared with the wild type ALG3, the relative activities of the mutant proteins I70T, Y72R, G98R, L157K, R171Q and M221T were 2.8%, 4.9%, 4.5%, 17.2%, 4.8% and 22.3%, respectively. The corresponding mutation activity was significantly higher in the patients with survival longer than 1 year than that in the patients with survival less than 1 year (P=0.002). Conclusion · An in vitro quantitative method is established to measure the enzymatic activities of ALG3-CDG related S. cerevisiae ALG3 mutants, providing the possibility to predict the severity of ALG3-CDG.

Key words: congenital disorder of glycosylation (CDG), mannosyltransferase, liquid chromatography tandem mass spectrometry (LC-MS), prokaryotic expression

中图分类号: