JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE) ›› 2020, Vol. 40 ›› Issue (11): 1461-1467.doi: 10.3969/j.issn.1674-8115.2020.11.004

• Original article (Basic research) • Previous Articles     Next Articles

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.

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

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