Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases.,ACS Central Science

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Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases.
ACS Central Science ( IF 12.7 ) Pub Date : 2020-02-19 , DOI: 10.1021/acscentsci.9b00021
Liang Lin _{1,

1} , Weston Kightlinger _{1,

1} , Sunaina Kiran Prabhu ₂ , Adam J Hockenberry ₁ , Chao Li ₂ , Lai-Xi Wang ₂ , Michael C Jewett _{1,

1} , Milan Mrksich _{1,

1,

1,

1}

Affiliation

Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to site-specifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.

中文翻译：

具有底物特异性N-糖基转移酶的蛋白质的顺序糖基化。

蛋白质糖基化是一种常见的翻译后修饰，会影响蛋白质的功能和特性。尽管通过单糖的化学酶促加工产生确定的糖蛋白的方法取得了进步，但是对糖蛋白的理解和工程化仍然具有挑战性，部分原因是难于在蛋白质的几个位置中的每个位点进行位点特异性控制糖基化。在这里，我们通过发现和利用N-糖基转移酶（NGT）的独特的，有条件的正交肽受体特异性来解决这一局限。我们使用无细胞蛋白质合成和自组装单分子层质谱技术快速筛选了41种假定的NGT，并使用1254个受体肽和8306个反应条件严格表征了四个NGT变体的独特受体序列偏好。然后，我们使用优化的NGT受体序列对将单糖顺序安装在一个靶蛋白内的四个位点上。这种位点特异性控制N-连接单糖的安装以加工成各种功能性N-聚糖的策略克服了合成用于研究和治疗应用的确定糖蛋白的主要限制。

更新日期：2020-02-26

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