Protein glycosylation, the post-translational attachment of complex sugar moieties (glycans), plays a crucial role in human health and is a critical quality attribute for protein therapeutic and vaccine production. However, our ability to rationally design and engineer glycoproteins with desired properties remains limited by a lack of high-throughput methods for synthesis and biochemical characterization of glycosyltransferases (GTs), the enzymes that attach glycans to proteins and then elaborate them. New tools are required to rapidly express polypeptide modifying GTs and determine design rules for engineering protein glycosylation sites which can be efficiently and specifically modified with a glycan of interest. Here we describe a systematic platform for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES) using cell-free protein synthesis and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. We synthesized six N- and O- linked polypeptide-modifying GTs from bacteria and humans and determined their sugar and peptide acceptor specificities at unprecedented depth and throughput using a total of 3,480 unique peptides and 13,903 unique reaction conditions. We then used cytoplasmic N-glycosyltransferase from Actinobacillus pleuropnemoniae (NGT) to demonstrate the use of GlycoSCORES to optimize and design polypeptide acceptor sequences in whole proteins. Optimization of NGT peptide substrates informed the design of small sequence motifs (GlycTags) that successfully directed efficient glycosylation onto the internal loops of three heterologous proteins, including a human immunogloblin (IgG1) Fc domain, in vitro and in the cytoplasm of living E. coli. We found peptide glycosylation levels accurately predict modification of whole proteins, enabling a 3-5 fold greater glycosylation efficiency for GlycTags compared to wild-type glycosylation sequences. This work provides a broadly applicable and systematic approach to facilitate the rational design of synthetic glycoproteins in bacteria and in vitro for compelling applications in glycoprotein therapeutics, vaccines, and diagnostics.

Design of protein glycosylation sites by high-throughput expression and characterization of polypeptide glycosyltransferases

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.