Characterization and subcellular localization of endomembrane proteases from tobacco BY-2 cells
Tobacco BY-2 suspension cells are used as a platform for production of valuable recombinant glycoproteins. The main drawback of plant cell cultures is the low expression level of biopharmaceuticals due to the presence of proteases. The recombinant biopharmaceuticals are subject to proteolysis by endogenous plant proteases while they travel through the secretory pathway, after secretion into the extracellular milieu or when mis-sorted to the vacuole. The degradation of recombinant proteins reduces the final yield and quality of the product. To increase recombinant glycoprotein production in tobacco BY-2 cells, a detailed understanding of the plant proteolytic machinery is necessary. The aim of this thesis was the biochemical characterization and subcellular localization of the three proteases previously cloned from tobacco BY-2 cells: cysteine (NtCP1), serine (NtSP1), and aspartic (NtAP1) proteases.To facilitate the detection and purification of aforementioned proteases, the C-termini have been fused to GFP (green fluorescent protein) and Strep II-tag. In vivo confocal microscopy findings revealed that theses proteases are targeted to the central vacuole. Recombinant proteases were purified from transgenic BY-2 cells via a anti-GFP antibodies coupled to magnetic beads or from transiently transformed N. benthamiana leaves via immobilized Strep-Tactin. In addition, proteolytic activity of these proteases was confirmed in vitro using artificial substrates. The maturation of NtAP1 was studied by mass spectrometry. From this study it was obvious that the NtAP1-GFP-Str converts to a mature form after deletion of propetide, part of PSI, and GFP. Beside this, in vitro analysis showed the purified NtAP1-GFP-Str can degrade pharmaceutical proteins used as model including M12 antibody and human serum albumin (HSA). Inhibitors and colocalization studies using endocytosis tracer FM4-64 revealed that these proteases deliver to the central vacuole via the classical route, which is composed of endoplasmic reticulum (ER), Golgi, trans-Golgi network/early endosome (TGN/EE) and pre-vacuolar compartment/late endosome (PVC/LE). Mutational analysis indicated the type of vacuolar sorting signal (VSS) within aforementioned proteases. The NtCP1 contains sequence specific vacuolar sorting signal (ssVSS), while the NtSP1 or NAP1 comprises C-terminal vacuolar sorting signal (ctVSS).These data showed that these vacuolar proteases may be involved in the degradation process of recombinantly produced biopharmaceuticals in tobacco BY-2 cells. From this study it seems that the degradation may occur in the first part of the secretory pathway.
Aachen, TH, Diss., 2015