Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases

Abstract

Glycosylation is an abundant post-translational modification that is important in disease and biotechnology. Current methods to understand and engineer glycosylation cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites modified by glycosyltransferases. Here we describe a systematic platform for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and mass spectrometry of self-assembled monolayers. We produced six N- and O-linked polypeptide-modifying glycosyltransferases from bacteria and humans in vitro and rigorously determined their substrate specificities using 3,480 unique peptides and 13,903 unique reaction conditions. We then used GlycoSCORES to optimize and design small glycosylation sequence motifs that directed efficient N-linked glycosylation in vitro and in the Escherichia coli cytoplasm for three heterologous proteins, including the human immunoglobulin Fc domain. We find that GlycoSCORES is a broadly applicable method to facilitate fundamental understanding of glycosyltransferases and engineer synthetic glycoproteins.

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

“…NGTs efficiently install a glucose residue onto asparagine side-chains within proteins using a uracil-diphosphate-glucose (UDP-Glc) sugar donor 24 . 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. First, the acceptor specificity of NGTs has been extensively studied using peptide and protein substrates 25,28 , glycoproteomic studies 25 , and the GlycoSCORES technique 26 . These studies revealed that NGTs modify N-X-S/T amino acid acceptor motifs resembling those in eukaryotic glycoproteins.…”

“…These studies revealed that NGTs modify N-X-S/T amino acid acceptor motifs resembling those in eukaryotic glycoproteins. Second, the NGT from Actinobacillus pleuropneumoniae (ApNGT) has been shown to modify both native and rationally designed glycosylation sites within eukaryotic proteins and peptides in vitro and in the E. coli cytoplasm 21,22,25,26 . 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 .…”

“…Complementing efforts in cellular engineering, E. coli-based cell-free protein synthesis (CFPS) systems can achieve gram per liter titers of diverse proteins in hours 30,32 and have been shown to enable the rapid discovery, prototyping, and optimization of biosynthetic pathways without the need to reengineer an organism for each pathway iteration 31,33,34 . By shifting the design-build-test unit from a genetic construct to a lysate, this approach allows for the construction of glycosylation pathways in hours [18][19][20]26 . We recently reported the development of a one-pot cellfree glycoprotein synthesis (CFGpS) system to synthesize glycoproteins in vitro by overexpressing glycan biosynthesis pathways and OSTs in the E. coli chassis strain before lysis 18 .…”

Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities

“…These examples include profiling lysine deacetylase (KDAC) specificities (Gurard-Levin, Kilian, Kim, Bahr, & Mrksich, 2010; Gurard-Levin, Kim, & Mrksich, 2009), as well as the substrate specificity of the protease OmpT (Wood et al, 2017). Additionally, we have used SAMDI and peptide arrays to optimize substrates for glycosyl transferases, and we have used carbohydrate arrays to discover new glycosyl transferase activities (Ban et al, 2012; Kightlinger et al, 2018). SAMDI peptide arrays are also compatible with measuring enzyme activities in cell lysates, as shown in a study observing distinct profiles of deacetylase activity associated with differentiation (Kuo, DeLuca, Miller, & Mrksich, 2013).…”