人源Alg1蛋白的原核表达及其跨膜结构域性质分析

Prokaryotic expression of human Alg1 protein and analysis of the transmembrane domain properties

摘要:

作为最普遍的蛋白质糖基化修饰方式，N-糖基化起始于内质网中多萜醇寡糖(dolichol-linked oligosaccharide,DLO)前体的合成。甘露糖基转移酶Alg1催化添加DLO结构中的第1个甘露糖，是该生化途径中的关键酶。人体中ALG1基因的缺陷会导致先天性糖基化疾病(congenital disorders of glycosylation,CDG)。为了建立人源Alg1(Homo sapiens Alg1,HsAlg1)的体外表达和活性检测体系，本研究构建了全长的pET28a-His6-HsAlg1和截除跨膜结构域(transmembrane domain,TMD)的pET28a-His6-HsAlg1₂₃₋₄₆₄这2种质粒并在大肠杆菌中表达，以多萜醇磷酸壳二糖(dolichyl-pyrophosphate GlcNAc2,DPGn2)为反应底物，采用液质联用的方法检测HsAlg1和HsAlg1₂₃₋₄₆₄重组蛋白的活性。结果显示，HsAlg1具有转糖基活性，蛋白纯化后活性降低，重新加入膜组分后得到部分回补，而HsAlg1₂₃₋₄₆₄不能催化转糖基反应。上述结果表明HsAlg1蛋白的N端TMD在催化反应中起到了重要的作用。本研究为后续表达并检测ALG1-CDG相关突变蛋白的活性奠定了基础。

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.