Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of DmPglB provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.