Liang Lin
Northwestern University, IL 60208, USA
Title: SITE-SPECIFIC GLYCOSYLATION OF PROTEINS VIA N-GLYCOSYLTRANSFERACES WITH SUBSTRATE ORTHOGONALITY
Biography
Biography: Liang Lin
Abstract
Glycosylation is the most complex post-translational modification and modulates the functions of many proteins. Glycoproteins isolated from living cells are generally a heterogeneous mixture of glycoforms, making it difficult to understand how specific glycan modifications effect protein function. Two-step chemoenzymatic glycosylation approach introduces glycoforms by first truncating the heterogeneous glycoforms into a monosaccharide primer and then using glycosyltransferases or glycosidases to elaborate the desired glycoform. However, this method lacks control in directing the initial glycosylation and therefore is typically not suited to preparing proteins having multiple, site-specifically defined oligosaccharide modifications. Here we introduce orthogonal N-glycosyltransferases (NGTs) to selectively direct the initial monosaccharide modification to a specific sequence in the protein. To identify the orthogonal NGTs, we developed an approach for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and high throughput screening with self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. We screened 41 putative NGTs and one reported NGT mutant and found four NGTs having orthogonal peptide substrate specificities. To further refine the specificities of NGTs, we prepared and characterized hundreds of NGT mutants and found sets of engineered mutants that have even greater orthogonality for peptide substrates. We then engineered the model protein colicin E7 immunity proteins with the four orthogonal sequences and sequentially treated the protein with the four corresponding NGTs to give a tetraglycosylated protein with 62% purity. Importantly, the hundreds of NGTs, forming an NGT library, that we characterized now provide the ability to modify most canonical N-glycosylation sequences, N-X-S/T (X is not P), and some non-canonical sequences, N-X-Z (Z is not S/T). Therefore, we can glycosylate approved or potential therapeutic proteins and vaccines (even those not containing canonical glycosylation sequences) using suitable NGTs from the NGT library, with site selectivity, to obtain desired properties such as prolonged serum life time and increased immunogenicity.