1 research outputs found
Exploring Site-Specific N‑Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes
The full potential of recombinant
Immunoglobulin A as therapeutic
antibody is not fully explored, owing to the fact that structure–function
relationships of these extensively glycosylated proteins are not well
understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of
the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered <i>Nicotiana benthamiana</i> plants and in human HEK293-6E cells.
All three IgA isotypes were purified and subjected to biophysical
and biochemical characterization. While no differences in assembly,
antigen binding, and glycosylation occupancy were observed, both systems
vary tremendously in terms of glycan structures and heterogeneity
of glycosylation. Mass-spectrometric analysis of site-specific glycosylation
revealed that plant-produced IgAs carry mainly complex-type biantennary
N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous
N-glycans with high levels of sialylation, core-fucose, and the presence
of branched structures. The site-specific analysis revealed major
differences between the individual N-glycosylation sites of each IgA
subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived
IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from <i>N. benthamiana</i> displayed numerous plant-specific modifications.
Interestingly, a shift in unfolding of the CH2 domain of plant-produced
IgA toward lower temperatures can be observed with differential scanning
calorimetry, suggesting that distinct glycoforms affect the thermal
stability of IgAs