Prokaryotic expression of human Alg1 protein and analysis of the transmembrane domain properties.
- Author:
Dongzhi WEI
1
;
Zhenghui CHEN
1
;
Chundi WANG
1
;
Xiaodong GAO
1
;
Ning WANG
1
Author Information
- Publication Type:Journal Article
- Keywords: N-glycosylation; dolichol-linked oligosaccharide; mannosyltransferase Alg1; membrane protein
- MeSH: Humans; Escherichia coli/metabolism*; Mannosyltransferases/biosynthesis*; Glycosylation; Recombinant Proteins/metabolism*; Protein Domains
- From: Chinese Journal of Biotechnology 2025;41(4):1535-1546
- CountryChina
- Language:Chinese
- Abstract: As the most common type of protein glycosylation, N-glycosylation begins with the synthesis of the dolichol-linked oligosaccharide (DLO) precursor in the endoplasmic reticulum. The mannosyltransferase Alg1 catalyzes the addition of the first mannose molecule to DLO, serving as a key enzyme in this biochemical pathway. The defect of human ALG1 gene can lead to the congenital disorders of glycosylation (CDG), i.e., ALG1-CDG. Therefore, it is of great significance to establish the expression and activity assay system of Homo sapiens Alg1 (HsAlg1) in vitro. In this study, full-length plasmid pET28a-His6-HsAlg1 and transmembrane domain-lacking plasmid pET28a-His6-HsAlg123-464 were constructed and expressed in Escherichia coli, and the activity of recombinant HsAlg1 and HsAlg123-464 was measured by liquid chromatography tandem mass spectrometry (LC-MS) with dolichyl-pyrophosphate GlcNAc2 (DPGn2) as the substrate. The results showed that HsAlg1 had transglycosylation activity, while the activity decreased after protein purification, which was partially restored upon re-addition of membrane components. However, HsAlg123-464 was unable to catalyze glycosylation. The results indicate that the N-terminal transmembrane domain (TMD) of HsAlg1 plays an important role in the catalytic reaction. This study lays a foundation for further expression and activity analysis of ALG1-CDG-related mutants.
