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标题：Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins
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作者：Mingji Li ; Xiaolu Zheng ; Sudhanshu Shanker 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：39
DOI：10.1073/pnas.2107440118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance Asparagine-linked ( N-linked) protein glycosylation—the covalent attachment of complex sugars to the nitrogen atom in asparagine side chains—is the most widespread posttranslational modification to proteins and also the most complex. N-glycosylation affects a significant number of cellular proteins and can have profound effects on their most important attributes such as biological activity, chemical solubility, folding and stability, immunogenicity, and serum half-life. Accordingly, the strategic installation of glycans at naïve sites has become an attractive means for endowing proteins with advantageous biological and/or biophysical properties. Here, we describe a glycoprotein engineering strategy that enables systematic investigation of the structural and functional consequences of glycan installation at every position along a protein backbone and provides a new route to bespoke glycoproteins. 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.
关键词：englycoengineering;glycosylation;protein design and engineering;synthetic biology