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


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Li M., Zheng X., Shanker S., Jaroentomeechai T., Moeller T. D. , Hulbert S. W. , ...More

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol.118, no.39, 2021 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 118 Issue: 39
  • Publication Date: 2021
  • Doi Number: 10.1073/pnas.2107440118
  • Journal Name: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Agricultural & Environmental Science Database, Animal Behavior Abstracts, Aquatic Science & Fisheries Abstracts (ASFA), Artic & Antarctic Regions, BIOSIS, CAB Abstracts, Chemical Abstracts Core, EconLit, EMBASE, Food Science & Technology Abstracts, INSPEC, Linguistic Bibliography, MathSciNet, MEDLINE, Pollution Abstracts, Psycinfo, Public Affairs Index, Veterinary Science Database, zbMATH, DIALNET
  • Keywords: glycoengineering, glycosylation, protein design and engineering, synthetic biology, N-LINKED GLYCOSYLATION, IMMUNITY-PROTEIN, CRYSTAL-STRUCTURE, RIBONUCLEASE-A, IM7, STABILITY, IMPROVES, PEPTIDE, PATHWAY, BINDING
  • Hacettepe University Affiliated: No

Abstract

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 antiHER2 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.