Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins.

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.
Competing Interests: Competing interest statement: M.P.D. has a financial interest in Glycobia, Inc., SwiftScale Biologics, Inc., and Versatope Therapeutics, Inc. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict of interest policies. J.J.G. is an unpaid board member of the Rosetta Commons. Under institutional participation agreements between the University of Washington, acting on behalf of the Rosetta Commons, Johns Hopkins University may be entitled to a portion of revenue received on licensing Rosetta software including some methods developed in this article. As a member of the Scientific Advisory Board, J.J.G. has a financial interest in Cyrus Biotechnology. Cyrus Biotechnology distributes the Rosetta software, which may include methods mentioned in this article. J.J.G.’s arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies.
(Copyright © 2021 the Author(s). Published by PNAS.)