Analysis of N. benthamiana β-galactosidases acting on glycoproteins
Abstract
Nicotiana benthamiana is one of the most widely used plant host to produce therapeutically relevant glycoproteins. However, production of recombinant proteins in plants may lead to the synthesis of aberrantly glycosylated proteins which can impair their biological activity. Outstanding achievements in glyco-engineering have been reported for N. benthamiana in the past. However, plant glyco-engineering must still overcome hurdles such as the correct subcellular expression of glyco-modulating enzymes and the adverse activity of endogenous plant glycosidases involved in trimming of complex N-glycans. For example, efficient β1,4-galactosylation was only achieved upon correctly targeting the β1,4-galactosyltransferase to the trans Golgi cisternae and tightly controlling its expression levels. Although these approaches succeeded in the galactosylation of monoclonal antibodies, it cannot be applied to many other recombinant proteins accumulating in the apoplast because of a specific removal of terminal β-galactosyl residues. We hypothesize that glycosyl-hydrolases, most probably β-galactosidases (BGAL), present in the plant secretome are responsible for the removal of non-reducing β1,4-galactose residues. The current project aims to characterize these two N. benthamiana BGALs by heterologous expression in insect cells, enzyme activity characterization, subcellular localization studies and N-glycan analysis. Furthermore, activity-based protein profiling and mass spectrometry will be carried out to identify additional BGALs that are less abundant or induced upon expression of the recombinant glycoprotein and the 1,4-galactosyltransferase. Modulation of the expression of relevant BGALs will enable the creation of a galactosidase-depleted environment in N. benthamiana that is highly suitable for the generation of recombinant glycoproteins with defined glycan structures.
Publikationen
Project staff
Alexandra Machado Ferreira Castilho
Dr. Alexandra Machado Ferreira Castilho
alexandra.castilho@boku.ac.at
Tel: +43 1 47654-94353
Project Leader
01.05.2018 - 31.10.2021