多萜醇磷酸β-葡萄糖基转移酶的活性和底物特异性分析

Analysis of enzyme activity and substrate specificity of dolichyl-phosphate b-glucosyltransferase

摘要:

内质网(endoplasmic reticulum, ER)是蛋白质折叠和质量控制的主要场所，其内部驻留的分子伴侣能够帮助新生多肽链形成正确的三级结构。部分分子伴侣通过特异性识别葡萄糖基化的N-寡糖结构与糖肽结合，进而促进相应蛋白的折叠。作为ER腔内葡萄糖基化反应的糖基供体，体外获取多萜醇磷酸葡萄糖(dolichol phosphate glucose, Dol-P-Glc)或其类似物对解析N-寡糖生物合成途径及糖蛋白质量控制体系具有重要意义，一直受到科学家们的关注。本研究以化学合成的一系列多萜醇(dolichol)类似物作为底物，对大肠杆菌表达的阴道毛滴虫(Trichomonas vaginalis)来源的多萜醇磷酸β-葡萄糖基转移酶E (dolichyl-phosphate b-glucosyltransferase E, Alg5E)进行了研究。结果表明该重组蛋白在体外具有较强的催化活性，可以识别不同链长的脂肪醇，底物中脂肪醇碳链越长其酶促反应进程越快，且对脂肪醇中异戊二烯的支链甲基表现出偏好性。研究还确认了重要的天冬氨酸-任意氨基酸-天冬氨酸(aspartate-any residue-aspartate, DXD)基序与二价金属离子的结合是该酶发挥催化功能的关键。上述工作为多萜醇寡糖(dolichol-linked oligosaccharide, DLO)合成途径中葡萄糖基转移酶Alg6、Alg8和Alg10的研究打下了基础。

Abstract:

Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated N-glycan moieties on peptide. It is of great significance to study the N-glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues in vitro. In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate b-glucosyltransferase E (Alg5E) derived from Trichomonas vaginalis. The results demonstrated that the recombinant Alg5E, expressed in Escherichia coli, exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme’s catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme’s catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).