Abstract
The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.
Highlights
THE IMMUNE SYSTEM The human immune system is an intricate system that serves to protect the body from invading organisms, prevent them from replicating or spreading and to remove them
In this study we considered the glycosylation pattern of immunoglobulin G (IgG) antibodies from pregnant women against human platelet antigens (HPA) of the fetus
We have focused on label-free analysis, which again can be done using area under the curve (AUC) or signal intensity based on precursor ions, or by counting the number of peptides assigned to a protein in an MS/MS experiment175
Summary
THE IMMUNE SYSTEM The human immune system is an intricate system that serves to protect the body from invading organisms, prevent them from replicating or spreading and to remove them. This anti-inflammatory effect of IVIg seems dependent on the presence of sialic acid at the Fc glycosylation site[3] Galactose is another carbohydrate of interest, and the amount of antibodies with galactose changes during the life cycle and during disease states, such as Chrohn’s disease or rheumatoid arthritis. Fucose on the other hand appears to have an essential role in antibody dependent cellular cytotoxicity (ADCC) and opsonophagocytosis as monoclonal humanized IgG1 with low levels of corefucose shows dramatic higher ADCC and opsonophagocytosis activity compared to the same antibodies with a higher amount of core-fucose [48] This is due to an increased affinity to FcɶRIIIa and FcɶRIIIb, respectively[2,13,47]
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