High-resolution mapping of glycoprotein structure-activity relationships by shotgun scanning glycomutagenesis

Mingji Li, Xiaolu Zheng, Sudhanshu Shanker, Thapakorn Jaroentomeechai, Ilkay Koçer, Josef Byrne, Emily C. Cox, Jason W. Labonte, Jeffrey J. Gray, Matthew P. DeLisa

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
JournalUnknown Journal
StatePublished - Jun 29 2020

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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