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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (6): 26-33    DOI: 10.13523/j.cb.20180604
研究报告     
酵母来源α-1,2甘露糖转移酶Alg11的异源表达、纯化和活性分析 *
李庆猛,李盛陶,王宁,高晓冬()
江南大学生物工程学院 糖化学与生物技术教育部重点实验室 无锡 214122
Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11
Qing-meng LI,Sheng-tao LI,Ning WANG,Xiao-dong GAO()
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology,Jiangnan University, Wuxi 214122, China
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摘要：

糖基转移酶Alg11作为N-糖基化途径中一个重要蛋白,功能为催化将甘露糖转移到底物DPGn2M3(Dolichyl-pyrophosphate-GlcNAc2Mannose3)上进而生成DPGn2M4和DPGn2M5这两种多萜醇寡糖前体的反应。在本研究中,首先通过对酿酒酵母Alg11的蛋白质结构进行分析,设计了去除跨膜域的蛋白Alg1145-548并成功在大肠杆菌中表达,进而对诱导时间、诱导剂浓度进行了产量最大化的优化,最终得到了纯化蛋白。以PPGn2(Phytanyl-pyrophosphate-GlcNAc2)为底物,利用体外表达的重组蛋白Alg1ΔTM和Trx-Alg2以酶法合成出Alg11的天然底物类似物PPGn2M3。用纯化的Alg1145-548蛋白催化转糖基反应,并通过液质联用(LC-MS)的方法检测产物,证实Alg1145-548蛋白具有催化PPGn2M3生成PPGn2M4和PPGn2M5的糖基转移酶活性。产物PPGn2M5通过不同的甘露糖苷酶酶切反应,验证了两个新加上的甘露糖以α-1,2糖苷键的形式连接到底物PPGn2M3上。底物特异性实验表明Alg1145-548可以特异性识别底物PPGn2M3,而其他N-糖基化中间体类似物如PPGn2和PPGn2M1无法被识别,且底物中的脂肪链结构也对酶的识别具有重要作用。Alg11的底物特异性保证了多萜醇寡糖前体的有序合成,具有重要的生理意义。
关键词： N-糖基化;  糖基转移酶Alg11;  液质联用(LC-MS);  诱导条件    
Abstract:

Glycosyltransferase Alg11, which is an important protein in N-glycosylation pathway, transfers the mannose moiety from GDP-Man to DPGn2M3 (Dolichyl-pyrophosphate-GlcNAc2Mannose3), forming DPGn2M4 and DPGn2M5 LLO (lipid-linked oligosaccharide) precursors. The structural analysis of Saccharomyces cerevisiae Alg11 showed the prediction of a hydrophobic N-terminal transmembrane domain. Thus, truncated Alg11 lacking the first 44 amino acid was designed and successfully overexpressed in Escherichia coli. The induction time and inducer concentration were optimized and the recombinant protein Alg1145-548 was purified. After the transferase activity assay, reaction mixture was applied to the liquid chromatography tandem mass spectrometry (LC-MS), which showed Alg1145-548 was capable to generate PPGn2M5 (Phytanyl-pyrophosphate-GlcNAc2Mannose5) from substrate PPGn2M3. Structural analysis of Gn2M5 showed the newly formed two glycosidic bonds could be cleaved by α-1,2 mannosidase, meaning the two mannose moieties were attached to Gn2M3 by α-1,2 linkages. Substrate specificity assay indicated the recombinant Alg11 specifically recognized PPGn2M3 rather than other LLOs, such as PPGn2 and PPGn2M1. Additionally, oligosaccharide Gn2M3 was not elongated by Alg1145-548, suggesting the lipid chain in the substrate PPGn2M3 was critical for the recognition. The achievement of active Alg11 provides an effective tool for producing Gn2M5, as well as for the further investigation of kinetic and mechanistic features of related mannosyltransferases.
Key words: N-glycosylation    Glycosyltransferase Alg11    Liquid chromatography tandem mass spectrometry (LC-MS)    Induction condition
收稿日期: 2018-01-23 出版日期: 2018-07-06
ZTFLH:  Q786  
基金资助: * 国家自然科学基金(21778023);江苏省自然科学基金(BK20170174);中央高校自主科研计划青年基金资助项目(JUSRP11727)
通讯作者: 高晓冬     E-mail: xdgao@jiangnan.edu.cn
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李庆猛
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引用本文:

李庆猛,李盛陶,王宁,高晓冬. 酵母来源α-1,2甘露糖转移酶Alg11的异源表达、纯化和活性分析 *[J]. 中国生物工程杂志, 2018, 38(6): 26-33.

Qing-meng LI,Sheng-tao LI,Ning WANG,Xiao-dong GAO. Expression, Purification and Activity Assay of Yeast α-1,2 Mannosyltransferase Alg11. China Biotechnology, 2018, 38(6): 26-33.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180604    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I6/26

图1  Alg11蛋白质跨膜域结构分析
图2  表达质粒pET28a-Alg1145-548的构建
图3  诱导时间(a)和IPTG浓度(b)对目的蛋白表达的影响
图4  全细胞和纯化后蛋白质电泳图(a)和Western blot验证图(b)
图5  重组蛋白活性分析及产物检测
图6  甘露糖苷酶验证产物
图7  Alg1145-548的底物特异性
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