TY - JOUR T1 - High-resolution mapping of glycoprotein structure–activity relationships by shotgun scanning glycomutagenesis JF - bioRxiv DO - 10.1101/2020.06.28.176198 SP - 2020.06.28.176198 AU - Mingji Li AU - Xiaolu Zheng AU - Sudhanshu Shanker AU - Thapakorn Jaroentomeechai AU - Ilkay Koçer AU - Josef Byrne AU - Emily C. Cox AU - Jason W. Labonte AU - Jeffrey J. Gray AU - Matthew P. DeLisa Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/06/29/2020.06.28.176198.abstract N2 - N-linked glycosylation serves to diversify the proteome and is crucial for the folding and activity of numerous cellular proteins. Consequently, there is great interest in uncovering the rules that govern how glycosylation modulates protein properties so that the effects of site-specific glycosylation might eventually be predicted. Towards this goal, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis (SSGM) that enables systematic investigation of the structural and functional consequences of glycan installation 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 a 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 the N-linked glycan in a manner that critically depended on the precise location of the modification. Comparison of the results with calculations of simple geometrics and Rosetta energies reveals nuanced mechanisms whereby glycosylation can affect protein activity. By enabling high-resolution mapping of glycan-mediated effects on acceptor proteins, glycomutagenesis opens up possibilities for accessing unexplored regions of glycoprotein structural space and engineering protein variants with advantageous biophysical and biological properties.Competing Interest StatementM.P.D. has a financial interest in Glycobia, Inc. and Versatope, 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 programs described here. 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 described in this paper. All other authors declare no competing interests. ER -