Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes.
J Proteome Res
; 16(7): 2560-2570, 2017 07 07.
Article
em En
| MEDLINE
| ID: mdl-28516782
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, whereas IgA1 from N. benthamiana 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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Isotipos de Imunoglobulinas
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Imunoglobulina A
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Receptor ErbB-2
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Trastuzumab
Limite:
Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Áustria