Chemoenzymatic Fc Glycosylation via Engineered Aldehyde Tags

Abstract

Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that was efficiently converted by recombinant formylglycine generating enzyme in vitro, thereby introducing aldehyde groups for subsequent chemical elaboration. Oxime-linked glycoconjugates were synthesized by conjugating aminooxy N-acetylglucosamine to the modified Fc followed by enzymatic transfer of complex N-glycans from corresponding glycan oxazolines by an EndoS-derived glycosynthase. In this manner we generated specific Fc glycoforms without relying on natural protein glycosylation machineries.

Piperidine-based glycodendrons as protein N-glycan prosthetics

“…25,26,39,40 Furthermore, we have previously generated a human IgG F c fragment bearing a CxPxR motif at the endogenous N-glycan site which could be conjugated with small aminooxy glycan moieties. 41 Reaction of aminooxy dendrimer 3 with the aldehyde-tagged F c fragment was performed at 60 ºC for 16 h in acetate buffer, pH 4. Analysis of the product by lectin blot demonstrated attachment of the Gal dendrimer to the human protein (Fig.…”

“…The Fc construct was expressed and purified according to Smith et al . 41 Briefly, CHO cells were transiently transfected with the Fc-containing plasmid, and the media was collected two days after transfection. After concentration, the aldehyde-tagged Fc was purified using a Protein A/G agarose resin (Pierce).…”

Protein–polymer therapeutics: a macromolecular perspective

“…This engineered aldehyde group thus allowed further conjugation with aminooxy N-acetylglucosamine and further oligosaccharide elongation with an engineered glycosynthase. 62 These sequential enzymatic reactions would be especially attractive when highly specific and efficient glycosylation is required on E. coli expressed proteins. Recently, recombinant approaches to achieve glycopolymer-based therapeutic proteins have even been marketed.…”

Recent Advances in Chemoenzymatic Bioconjugation Methods

“…Conjugation with an aminooxy GlcNAc enabled subsequent elaboration of the glycan with oxazoline glycans catalyzed by the EndoS mutant D233Q , providing a fully synthetic glycan. 66 Canonically, an oxime formed with the aldehyde moiety of fGly is an efficient bioconjugation strategy, but our laboratory has found this particular oxime to be hydrolytically unstable. To circumvent this problem, Agarwal, Kudirka and coworkers have developed new chemistries that form stable bonds with fGly, based on the Pictet-Spengler reaction 67 and the Knoevenagel reaction.…”