Design of Protein Glycosylation Sites By Cell-Free Protein Synthesis and Mass Spectrometry of Self-Assembled Monolayers 

Kightlinger, W., Northwestern University
Lin, L., Northwestern University
Mrksich, M., Northwestern University
Jewett, M. C., Northwestern University
Protein glycosylation, the post-translational attachment of complex oligosaccharides (glycans), is the most abundant polypeptide modification in eukaryotes, and is integrally involved in human biology and disease. Changes in glycosylation have been shown to modulate the pharmacokinetics and potency of protein therapeutics and vaccines, making them critical quality attributes for biotechnology applications. However, the study of glycans in native systems (glycobiology) and the intentional manipulation of protein glycosylation patterns (glycoengineering) remain limited by a shortage of methods to characterize glycosylation enzyme activities and a reliance on endogenous glycosylation systems which can produce highly variable glycosylation patterns and constrain choices of production hosts, protein trafficking, and possible glycoforms. The structural complexity of enzymes involved in initial modification of polypeptides with glycans and the vast sequence spaces which must be surveyed to determine their specificities have impeded efforts to implement synthetic glycosylation systems in heterologous production hosts, such as Escherichia coli. New tools are required to rapidly express polypeptide modifying glycosyltransferases and determine design rules for engineering protein glycosylation sites which can be efficiently and specifically modified with a glycan of interest.

Here we describe an in vitro platform for high-throughput expression and characterization of glycosylation enzymes using E. coli Cell-Free Protein Synthesis (CFPS) and Self-Assembled Monolayers for Desorption Ionization Mass Spectrometry (SAMDI-MS). This workflow allowed us to produce >800 µg/mL of a cytoplasmic N-linked glycosyltransferase (NGT) in vitro and determine its peptide acceptor and sugar donor specificities at unprecedented depth and throughput with ~10,000 unique reactions conditions and ~3,000 unique peptide substrates. We used this dataset to develop a small, robust acceptor sequence motif (GlycTag) to direct the efficient installation of N-linked glycans onto the internal loops of heterologous protein substrates in vitro and in the cytoplasm of living E. coli. We found that SAMDI-MS observations at the peptide level accurately predicted modification trends in the context of whole proteins. We placed GlycTags in three structurally diverse target proteins, including the Fc region of a human antibody, and found that these sequences were modified at 3-5 fold greater efficiency compared to wild-type glycosylation sequence motifs. Notably, this N-linked glycosylation system does not require protein transport across cellular membranes or the use of membrane bound components, making it particularly attractive for the development of synthetic glycosylation systems.

This work provides a disruptive technique for glycosyltransferase characterization and provides a new set of tools towards emerging bacterial and in vitro glycosylation platforms. Ultimately, these glycoengineering efforts will enable a deeper understanding of glycan structure and function and help to bring about a new generation of rationally designed glycoprotein therapeutics, vaccines, and diagnostics.