GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins

Abstract

Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azidosialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and coppercatalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation

“…We therefore sought to produce one of these trisaccharides using a completely in vitro enzymatic approach. LgtB, a glycosyltransferase from Neisseria meningitidis, has been shown to install galactose onto GlcNAc in a β-1,4 linkage, and PdST6, a glycosyltransferase from Photobacterium damselae, has been shown to install sialic acid in an α-2,6 linkage. ,, We incorporated these enzymes and the appropriate nucleotide-activated sugar donors, uridine diphosphate α-D-galactose (UDP- galactose) and cytidine monophosphate sialic acid (CMP-sialic acid), into our reaction solution to perform elaboration to a sialylated N -acetyllactosamine (sialyl-LacNAc) structure.…”

“…coli, these lysates offer a blank slate for constructing glycosylation pathways. To date, cell-free systems have been used to produce conjugate vaccines − and sialoglycoproteins as well as understand glycosyltransferase acceptor sequence preference and prototype protein glycosylation pathways . To the best of our knowledge, however, an integrated system for the in vitro synthesis of both the acceptor protein and an undecaprenyl phosphate–linked GlcNAc followed by the subsequent enzymatic glycosylation of the acceptor protein with a single GlcNAc residue has not been reported.…”

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

“…To ensure that the AlphaLISA binding signal was allergen-specific and not confounded by possible contaminants in the purified reaction, we performed a similar cotitration where Der p 2 was modified with a trisaccharide to block the epitope . Installing the trisaccharide abrogated IgE binding, indicating that binding only occurs when the Der p 2 epitope is present and accessible (Figure S2).…”