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Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins [Applied Biological Sciences]
Proceedings of the National Academy of Sciences of the United States of America ( IF 11.1 ) Pub Date : 2021-09-28 , DOI: 10.1073/pnas.2107440118
Mingji Li 1 , Xiaolu Zheng 1 , Sudhanshu Shanker 2 , Thapakorn Jaroentomeechai 1 , Tyler D Moeller 1 , Sophia W Hulbert 3 , Ilkay Koçer 1 , Josef Byrne 1 , Emily C Cox 4 , Qin Fu 5 , Sheng Zhang 5 , Jason W Labonte 2, 6 , Jeffrey J Gray 7 , Matthew P DeLisa 3, 4, 5, 8
Affiliation  

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein–protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.



中文翻译:

鸟枪扫描糖突变:构建和表征新糖蛋白的简单有效策略 [应用生物科学]

作为一种常见的蛋白质修饰,天冬酰胺连接的 ( N-链接) 糖基化有能力极大地影响蛋白质的生物学和生物物理特性。然而,糖基化作为具有优势特性的工程蛋白质的策略的常规使用受到我们无法构建和筛选大量糖蛋白以对聚糖安装的后果进行编目的限制。为了应对这一挑战,我们描述了一种称为鸟枪扫描糖突变的组合策略,其中构建了编码给定蛋白质所有可能的糖基化位点变体的 DNA 文库,然后在具有糖基化能力的细菌中表达,从而能够快速确定蛋白质中的可糖基化位点。由此产生的新糖蛋白可以很容易地进行可用的高通量检测,使得系统地研究沿着蛋白质骨架的聚糖缀合的结构和功能后果成为可能。三种不同的受体蛋白证明了这种方法的效用,即细菌免疫蛋白 Im7、牛胰核糖核酸酶 A 和人抗 HER2 单链 Fv 抗体,所有这些都被发现耐受N-聚糖附着在大量位置且效率相对较高。许多糖变体的稳定性和活性被N-连接聚糖以一种严重依赖于修饰的精确位置的方式显着改变。结构模型表明,通过在蛋白质-蛋白质界面的外围与聚糖建立新的界面接触,可以提高亲和力。重要的是,我们预计我们的糖突变工作流程应该提供对糖蛋白结构空间的未探索区域和具有理想特性的定制新糖蛋白的访问。

更新日期:2021-09-23
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