Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes

Journal of Proteome Research
Kathrin GöritzerRichard Strasser

Abstract

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 Nicotiana benthamiana 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, wh...Continue Reading

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Citations

Aug 15, 2019·Biological Reviews of the Cambridge Philosophical Society·Harry C TjondroMorten Thaysen-Andersen
Aug 3, 2019·International Journal of Molecular Sciences·Nicolas SmargiassoCatherine Navarre
Aug 1, 2019·The Journal of Biological Chemistry·Kathrin GöritzerRichard Strasser
Dec 11, 2019·Antibodies·Patrícia de Sousa-Pereira, Jenny M Woof
Dec 19, 2020·Antibodies·Geert van TeteringJeanette Leusen
Aug 8, 2019·The World Allergy Organization Journal·Josef SingerErika Jensen-Jarolim
Mar 11, 2020·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Huimin LiuDingkun Zhang
Jul 30, 2021·Plant Methods·Izabela Anna Chincinska
Aug 21, 2020·Trends in Biotechnology·Emmanuel A MargolinAnn E Meyers

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Methods Mentioned

BETA
glycosylation
PCR
transfection
size-exclusion chromatography
Light
differential scanning calorimetry
reversed-phase chromatography
ELISA

Software Mentioned

ProGlycAn
ProteinScape
Data Analysis
Origin
MASCOT
DataAnalysis

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