Expression and characterization of antibodies directed to vacuoles in leaves of Nicotiana benthamiana

OCAMPO,CAROLINA GABRIELA; LAREU, FABRICIO; PETRUCCELLI, SILVANA

Iguazú Falls

Congreso; International Plant Molecular Biology Congress (IPMB); 2015

International Society for Plant Molecular Biology (ISPMB)

Plants are one of the most economical plantform for large-scale production of therapeutic proteins. The majority of this proteins are directed to the secretory pathway. However, to generate highly active molecules a detailed understanding of biochemical and cell biological processes that affect protein stability and post-translational modifications is required. In this work we aimed to contribute with the knowledge of this route and examine vacuolar deposition as a possible strategy for the expression of mAb. The mAb 14D9 was transiently expressed in Nicotiana benthamiana leaves in: a secreted form; two different vacuolar forms and an ER-retained form. All the versions of 14D9 were purified, vacuolar antibody and ER-retained antibodies proteins yields were similars. Vacuolar versions were subjected to MS analyses to determine the N-glycosylation profile. Three major glycoforms were detected: Man7, Man8 as well as fucosylated (F) and xylosylated (X) structures. To monitor subcellular localization secretory heavy (H) and light (L) chains were fused to RFP. Confocal microscopy showed that sec-L-RFP localized in the apoplast, while sec-H-RFP was retained in the ER. When sec-L-RFP and H-CtVSS or H-ssVSS or sec-L and H-RFP-CtVSS were co-infiltrated, red fluorescence was observed in the central vacuole, no ER staining was found.Vacuolar targeted 14D9 accumulates in the central vacuole of leaves, thus we can conclude that Man7 and Man8, ER typical glycosylation, indicates a direct transport from the ER to the vacuole bypassing the Golgi. However, at least some of the antibody molecules pass through the trans-Golgi as shown by FX containing glycans.