Chemoenzymatic Fc glycosylation via engineered aldehyde tags

Bioconjug Chem. 2014 Apr 16;25(4):788-95. doi: 10.1021/bc500061s. Epub 2014 Apr 7.


Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that was efficiently converted by recombinant formylglycine generating enzyme in vitro, thereby introducing aldehyde groups for subsequent chemical elaboration. Oxime-linked glycoconjugates were synthesized by conjugating aminooxy N-acetylglucosamine to the modified Fc followed by enzymatic transfer of complex N-glycans from corresponding glycan oxazolines by an EndoS-derived glycosynthase. In this manner we generated specific Fc glycoforms without relying on natural protein glycosylation machineries.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aldehydes / chemistry
  • Aldehydes / metabolism*
  • Animals
  • CHO Cells
  • Cricetulus
  • Glycosylation
  • Immunoglobulin Fc Fragments / chemistry*
  • Immunoglobulin Fc Fragments / genetics
  • Immunoglobulin Fc Fragments / metabolism*
  • Immunoglobulin G / chemistry
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / metabolism*
  • Protein Engineering*
  • Recombinant Proteins / metabolism
  • Streptococcus pyogenes / enzymology


  • Aldehydes
  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Recombinant Proteins
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase