Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

ACS Chem Biol. 2017 Jun 16;12(6):1665-1673. doi: 10.1021/acschembio.7b00282. Epub 2017 May 5.

Abstract

The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans.

Publication types

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

MeSH terms

  • Asparagine / metabolism
  • Cell Engineering / methods*
  • Erythropoietin / metabolism*
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Polysaccharides / metabolism*
  • Protein Engineering / methods*
  • Tandem Mass Spectrometry

Substances

  • Polysaccharides
  • Erythropoietin
  • Asparagine