In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn 297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn 297 , either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn 297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn 221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

This information is current as Viruses Human IgG1 Inhibitors of Influenza B Functional Glycosylation of the Fc Region of Choice of Host Cell Line Is Essential for the and Richard J. Pleass

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in α2,6-linkage (α2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.