A KDEL tagged monoclonal antibody is efficiently retained in the reticulum endoplasmic in leaves while is both partially secreted and sorted to protein storage vacuoles in seeds

SILVANA PETRUCCELLI*, MARISA OTEGUI, FABRICIO LAREU, OLIVIA TRANDINHTHANHLIEN, ANNE-CATHERINE FITCHETTE-LAINE, ARIANA CIRCOSTA, MARTIN RUMBO, MURIEL BARDOR , ROSA CARCAMO, VERONIQUE GOMORD, ROGER N. BEACHY

Plant Biotechnology Journal

Blackwell Publishing

Lugar: Bristol; Año: 2006 vol. 4 p. 511 - 527

1467-7652

Transgenic plants are attractive biological systems for the large-scale production of pharmaceutical proteins. In particular, seeds offer special advantages, such as easy handling and long term, stable storage. Nevertheless, most of the studies of expression of antibodies in plants have been performed in leaves.  We now report the expression of a secreted (sec-Ab) or KDEL-tagged mutant (Ab-KDEL) of the 14D9 monoclonal antibody in transgenic tobacco leaves and seeds. While the KDEL sequence has little effect on the accumulation of the antibody in leaves, it leads to a higher antibody yield in seeds. Sec-AbLeaf purified from leaf contains complex N-glycans, including Lewisa epitopes, as typically found in extracellular glycoproteins. In contrast, Ab-KDEL Leaf bears only high mannose type oligosaccharides (mostly Man 7 and 8) consistent with an efficient ER retention/cis-Golgi retrieval of the antibody. Sec-Ab and Ab-KDEL gamma chains purified from seeds are cleaved by proteases and contains complex N-glycans indicating maturation in the late Golgi compartments. Consistent with glycosylation of the protein, the Ab-KDELSeed was partially secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER. This dual targeting may be due to KDEL-mediated targeting to the PSV and to a partial saturation of the vacuolar sorting machinery. Taken together, our results point out important differences in the ER retention and vacuolar sorting machineries between leaves and seeds. In addition, we demonstrate that a plant-made antibody with triantennary high-mannose type N-glycans has similar Fab functionality as compared to its counterpart having biantennary complex N-glycans but the former antibody interacts with protein A in a stronger manner and is more immunogenic than the latter. Such differences could be related to a variable IgG-Fc folding that would depend on the size of the N-glycan