Transport and deposition of vacuolar antibodies in Nicotiana benthamiana leaves

OCAMPO CAROLINA GABRIELA; LAREU FABRICIO; PETRUCCELLI SILVANA

Foz do Iguazu

Congreso; 11th International Congress of Plant Molecular Biology; 2015

Plants are the most economical platform for large-scale production of therapeutic proteins. The final destination of secretory proteins is the apoplast, where an intense proteolysis occurs. In this work we aimed to examine vacuolar deposition as a possible strategy for the expression of monoclonal antibodies (mAb) and also clarify the transit route to the vacuole.Different versions of the mAb 14D9: secretory (sec-), reticulum endoplasmic retained (ER-) and two vacuolar forms (CtVSS and ssVSS) were transiently expressed in Nicotiana benthamiana leaves. Five days after infiltration all the versions of 14D9 were purified by affinity chromatography and the amount of purified antibodies were quantified. Vac-Abs and ER-Ab proteins yields were similars and higher than sec-Ab ones. Vac-Abs 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.