Expression of Antibody Fragments with a Controlled N-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

Author:

Loos Andreas1,Van Droogenbroeck Bart1,Hillmer Stefan1,Grass Josephine1,Pabst Martin1,Castilho Alexandra1,Kunert Renate1,Liang Mifang1,Arcalis Elsa1,Robinson David G.1,Depicker Ann1,Steinkellner Herta1

Affiliation:

1. Department of Applied Genetics and Cell Biology (A.L., A.C., E.A., H.S.), Department of Chemistry (J.G., M.P.), and Department of Applied Microbiology (R.K.), University of Natural Resources and Applied Life Sciences, A–1190 Vienna, Austria; Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Plant Biotechnology and Genetics, Ghent University, B–9052 Ghent

Abstract

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.

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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