GLYCO-BUILD: an enzymatic pipeline for the synthesis of peptides carrying eukaryotic N-glycans

Peptides and proteins containing defined N-glycans hold great promise for biomedicine because the attached glycans engage in specific interactions, influence targeted delivery and folding, and improve pharmacokinetic properties. However, challenges associated with producing homogeneous N-glycoconjugates hinder their wider application. Here, we report the development of GLYCO-BUILD, an enzymatic pipeline that can generate glycopeptides carrying eukaryotic N-glycans at a level of homogeneity not accessible by alternative methodologies. The pipeline recapitulates the endoplasmic reticulum-based steps of the eukaryotic protein N-glycosylation machinery. However, it employs a combination of enzymes of archaeal, bacterial, and eukaryotic origin that are able to process inexpensive phytol instead of the native dolichol as a lipid carrier. GLYCO-BUILD facilitates the synthesis of homogeneous N-glycans ranging from GlcNAc₂ to GlcNAc₂Man₉Glc₃, and their transfer to acceptor polypeptides using single-subunit oligosaccharyltransferases (OSTs) from the eukaryotic parasite Trypanosoma brucei. We used GLYCO-BUILD to generate glycopeptides mimicking viral mannosylated antigens, glucosylated species and precursors of complex and hybrid glycans. Our pipeline is modular, versatile and can be combined with other approaches of glycan extension and modification to generate a wide range of homogeneous N-glycoconjugates for use in research, diagnostics and therapeutics, including serum testing, vaccine development or modulation of biotherapeutics' half-life.