A novel enzymatic method for synthesis of glycopeptides carrying natural eukaryotic N-glycans

Abstract

A novel enzymatic approach was developed for facile production of glycopeptides carrying natural eukaryotic N-glycans. In this approach, peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGT) and a glucosamine N-acetyltransferase (GlmA), respectively. The resulting GlcNAc-peptides were further modified by an endo-β-N-acetylglucosaminidase M mutant (EndoM) to generate glycopeptides. In three steps of enzymatic catalysis, glycopeptides carrying complex-type N-glycans can be efficiently synthesized.

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 …”

“… 170 A variation of this method using an engineered NGT (ApNGT Q469A ) to install GlcN along with an acetyltransferase (GlmA) enabled the synthesis of an authentic human N -linked glycopeptide with GlcNAc at the reducing end. 172 The discovery of NGT homologues with unique and conditionally orthogonal peptide acceptor specificities combined with transglycosylation strategies has recently enabled the sequential, site-specific installation of multiple distinct glycans on a single target protein. 83 While further efforts are needed to enhance efficiency of such an approach, this advances a new concept for synthesizing defined glycoproteins for research and therapeutic applications.…”

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

“…Synthetic NGT-based glycosylation systems are not limited by OST substrate specificities and do not require protein transport across membranes or lipid-associated components 9 . These systems have elicited great interest as a complementary approach for synthesis of glycoproteins, including therapeutics and vaccines, that are difficult or impossible to produce using OST-based systems 9,16,22,28,30–32 . Several recent advances set the stage for this vision.…”

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

“…Importantly, NGTs are soluble enzymes that can be easily expressed functionally in the E. coli cytoplasm 22,25,26 . Because glycosylation systems using NGT for glycan-protein conjugation do not require protein transport across membranes or lipid-associated components, they have elicited great interest from the glycoengineering community for the production of recombinant protein therapeutics and vaccines 9,21,22,[26][27][28][29] . Several recent advances set the stage for this vision.…”

“…Third, the Aebi group recently reported a biosynthetic method in E. coli cells for elaborating the N-linked glucose installed by ApNGT to a polysialic acid motif, which may prolong the serum-half-life of small proteins 21 . Chemoenzymatic methods to transfer pre-built, oxazoline-functionalized oligosaccharides onto the ApNGT-installed glucose residue have also been reported 27,29 . However, other biosynthetic pathways to build therapeutically relevant glycans using NGTs have not been explored 9 , leaving much of the vast theoretical utility of this enzyme class for biocatalysis inaccessible.…”