Site-Directed Glycosylation of Peptide/Protein with Homogeneous O-Linked Eukaryotic N-Glycans

Abstract

Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine α-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.

Production of Glycopeptide Derivatives for Exploring Substrate Specificity of Human OGA Toward Sugar Moiety

“…We previously evaluated chemoenzymatic synthesis and purified oligosaccharides and glycopeptides (Wen et al, 2016a,b; Wu et al, 2016; Wang et al, 2018). As an extension of these studies, glycopeptides containing different GlcNAc derivatives were successfully synthesized and purified.…”

Synthetic Glycobiology: Parts, Systems, and Applications

“… 238 , 239 (c) Chemoenzymatic methods have been developed to install full-length human glycans. Primary strategies include: (i) endoglycosidase-mediated transglycosylation 206 for remodeling glycans produced in yeast or CHO cells; (ii) enzymatic “tag and modify” approaches which use engineered bacteria or purified enzymes to install O -linked GlcNAc, 240 N -linked GlcNAc from an exoglycosidase-treated C. jejuni heptasaccharide, 241 N -linked Glc installed by NGT, 170 or an N -linked GlcNAc installed by NGT and acetyltransferase GlmA 172 which can then be elaborated to full-length N -linked glycans using transglycosylation; (iii) chemical “tag and modify” methods that directly modify cysteine or noncanonical amino acids within proteins to install glycan handles that can be further elaborated by transglycosylation; 242 − 244 and (iv) total chemical synthesis approaches that use solid phase-peptide synthesis to directly incorporate glycosylated amino acids into peptides which can then be linked together using native chemical ligation approaches. 25 , 41 , 245 …”

Chemoenzymatic Methods for the Synthesis of Glycoproteins

“…Recently, Peng George Wang and co-workers described an efficient site-directed glycosylation of peptide/protein with homogeneous O-linked N-glycans using OGT transferase coupled with an endoglycosidase mutant-catalyzed sugar chain extension. 404 It was achieved by transferring an O-GlcNAc to chemically synthesized peptide sequences, derived from bioactive proteins (Scheme 32a). The method was further expanded to an in vivo O-GlcNAc modification of a bovine protein, α -Crystallin mutant (Crys-A), by coexpressing OGT and Crys-A in E. coli and a subsequent N-glycosylation on the GlcNAc site using Endo-M N175Q in vitro (Scheme 32b).…”