(502e) Design of Glycosylation Sites By Rapid Synthesis and Analysis of Glycosyltransferases

Authors: 
Kightlinger, W., Northwestern University
Lin, L., Northwestern University
DeLisa, M. P., Cornell University
Jewett, M. C., Northwestern University
Mrksich, M., Northwestern University
Rosztoczy, M., Northwestern University
Li, W., Northwestern University

Protein glycosylation, the post-translational attachment
of oligosaccharides (glycans), is the most abundant polypeptide modification in
eukaryotes, and is integrally involved in human biology and
disease. Glycosylation is present in 70% of approved or preclinical protein
therapeutics and has profound effects on protein stability, immunogenicity, and
potency, motivating close study and intentional engineering of glycosylation
sites and structures. 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 test them with sufficient throughput 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 the cytoplasmic N-glycosyltransferase
from Actinobacillus pleuropnemoniae (NGT) to demonstrate the use of
GlycoSCORES for optimization and design of polypeptide acceptor sequences within
whole proteins.
Our optimization
of NGT peptide substrates resulted in the discovery of many new peptide
sequences with superior activity to those previously proposed or found in
nature. These winning peptide sequences 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.