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

Authors:

Belen Sotomayor, Thomas C. Donahue, Sai Pooja Mahajan, May N. Taw, Sophia W. Hulbert, Erik J. Bidstrup, D. Natasha Owitipana, Alexandra Pang, Xu Yang, Souvik Ghosal, Christopher A. Alabi, Parastoo Azadi, Jeffrey J. Gray, Michael C. Jewett, Lai-Xi Wang, Matthew P. DeLisa

Affiliation:

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.; Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA.; Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.; Cornell Institute of Biotechnology, Cornell University, 130 Biotechnology Building, Ithaca, NY 14853, USA.; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.; Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.; Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602-4712, USA.

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Description:

Human immunoglobulin G (IgG) antibodies are a major class of biotherapeutics and undergo N‑linked glycosylation in their Fc domain, which is critical for immune functions and therapeutic activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. Previous attempts to engineer Escherichia coli for this purpose have met limited success due in part to the lack of oligosaccharyltransferase (OST) enzymes that can install N‑glycans at the conserved N297 site in the Fc region. Here, we identify a single‑subunit OST from Desulfovibrio marinus with relaxed substrate specificity that catalyzes glycosylation of native Fc acceptor sites. By chemoenzymatic remodeling the attached bacterial glycans to homogeneous, asialo complex‑type G2 N‑glycans, the E. coli‑derived Fc binds human FcγRIIIa/CD16a, a key receptor for antibody‑dependent cellular cytotoxicity (ADCC). Overall, the discovery of D. marinus OST provides previously unavailable biocatalytic capabilities and sets the stage for using E. coli to produce fully human antibodies.

Publications:

Tags:

Chemical biology Glycosylation Modeling and Simulation Peptides and proteins

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