Abstract: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 the α-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.
