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Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis

(2011) PLANT PHYSIOLOGY. 155(4). p.2036-2048
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Abstract
Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.
Keywords
HUMAN MONOCLONAL-ANTIBODY, ENGINEERED ANTIBODIES, ESCHERICHIA-COLI, LC-ESI-MS, ANTI-ERBB2 IMMUNOAGENTS, HEPATITIS-A VIRUS, TRANSGENIC TOBACCO SEED, IMMUNODEFICIENCY-VIRUS TYPE-1, STORAGE VACUOLES, SINGLE-CHAIN ANTIBODIES

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MLA
Loos, Andreas, Bart Van Droogenbroeck, Stefan Hillmer, et al. “Expression of Antibody Fragments with a Controlled N-glycosylation Pattern and Induction of Endoplasmic Reticulum-derived Vesicles in Seeds of Arabidopsis.” PLANT PHYSIOLOGY 155.4 (2011): 2036–2048. Print.
APA
Loos, A., Van Droogenbroeck, B., Hillmer, S., Grass, J., Pabst, M., Castilho, A., Kunert, R., et al. (2011). Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. PLANT PHYSIOLOGY, 155(4), 2036–2048.
Chicago author-date
Loos, Andreas, Bart Van Droogenbroeck, Stefan Hillmer, Josephine Grass, Martin Pabst, Alexandra Castilho, Renate Kunert, et al. 2011. “Expression of Antibody Fragments with a Controlled N-glycosylation Pattern and Induction of Endoplasmic Reticulum-derived Vesicles in Seeds of Arabidopsis.” Plant Physiology 155 (4): 2036–2048.
Chicago author-date (all authors)
Loos, Andreas, Bart Van Droogenbroeck, Stefan Hillmer, Josephine Grass, Martin Pabst, Alexandra Castilho, Renate Kunert, Mifang Liang, Elsa Arcalis, David G Robinson, Anna Depicker, and Herta Steinkellner. 2011. “Expression of Antibody Fragments with a Controlled N-glycosylation Pattern and Induction of Endoplasmic Reticulum-derived Vesicles in Seeds of Arabidopsis.” Plant Physiology 155 (4): 2036–2048.
Vancouver
1.
Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, et al. Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. PLANT PHYSIOLOGY. 2011;155(4):2036–48.
IEEE
[1]
A. Loos et al., “Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis,” PLANT PHYSIOLOGY, vol. 155, no. 4, pp. 2036–2048, 2011.
@article{1213349,
  abstract     = {Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.},
  author       = {Loos, Andreas and Van Droogenbroeck, Bart and Hillmer, Stefan and Grass, Josephine and Pabst, Martin and Castilho, Alexandra and Kunert, Renate and Liang, Mifang and Arcalis, Elsa and Robinson, David G and Depicker, Anna and Steinkellner, Herta},
  issn         = {0032-0889},
  journal      = {PLANT PHYSIOLOGY},
  keywords     = {HUMAN MONOCLONAL-ANTIBODY,ENGINEERED ANTIBODIES,ESCHERICHIA-COLI,LC-ESI-MS,ANTI-ERBB2 IMMUNOAGENTS,HEPATITIS-A VIRUS,TRANSGENIC TOBACCO SEED,IMMUNODEFICIENCY-VIRUS TYPE-1,STORAGE VACUOLES,SINGLE-CHAIN ANTIBODIES},
  language     = {eng},
  number       = {4},
  pages        = {2036--2048},
  title        = {Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis},
  url          = {http://dx.doi.org/10.1104/pp.110.171330},
  volume       = {155},
  year         = {2011},
}

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