Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases

Abstract

Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to sitespecifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.

Semi-processive hyperglycosylation of adhesin by bacterial proteinN-glycosyltransferases

“…Moreover, especially the bacterial adhesins and autotransporters share a general β-helical fold, 44,45 which is also highly associated with two-partner secretion proteins in different species. 46,47 It will be highly revealing to investigate other known and predicted NGTs for processive characteristics, 48 of Glc-modifications on the HMW adhesins before export by the HMW1B translocator. In general, the density of epitopes is directly linked to the efficiency of natural multivalent interactions, and is proposed to serve as a mechanism to regulate the biological interaction.…”

“…Several studies have focused on employing NGTs (and their engineered variants) in the biosynthesis of defined glycoproteins for biotechnological applications and vaccine development. 38,48,[53][54][55] The ApNGT mutant Q469A showed reduced product inhibition, and produced a more homogenously glycosylated HMW1ct, with up to 10 residues. Based on the central position of Q469 in both UDP-Glc and peptide binding as revealed by molecular modeling, we propose that Q469 may function as a 'processive switch', preventing the glycosylated product from leaving the binding site, and thereby increasing the association required for an additional round of catalysis.…”

Synthetic Glycobiology: Parts, Systems, and Applications

“… 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.…”

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

“…More recently, the same team extended the methodology to the analysis of intact glycoproteins (Techner et al, 2020 ), providing an exciting new avenue for the discovery and improvement of glycosylation enzymes. In addition, they used conditionally orthogonal peptide acceptor specificities of NGTs to site-specifically control installation of multiple distinct glycans (Lin et al, 2020 ).…”