Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins

Abstract

Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the same recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.

The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins

“…Accordingly, KDEL-tagged recombinant proteins are often found at least partially in PSVs, e.g., a single chain antibody fragment (scFv) in rice (Torres et al, 2001) and HSA in wheat (Arcalis et al, 2004). The direct ER-to-PSV transport of recombinant proteins has also been reported in dicot seeds (Petruccelli et al, 2006; Floss et al, 2009; Loos et al, 2011a; Morandini et al, 2011; Arcalis et al, 2013). The endogenous KDEL-tagged proteinase sulfhydryl-endopeptidase (SH-EP) behaves in a similar manner in the cotyledons of germinating mung bean seeds (Toyooka et al, 2000; Okamoto et al, 2003), and storage protein precursors in pumpkin seeds are delivered to PSVs in precursor-accumulating (PAC) vesicles (Hara-Nishimura et al, 1998).…”

“…The lack of secretion in cereal endosperm often results in a distinct lack of typical apoplast N -glycan structures such as GnGnXF, which are abundant in proteins expressed in leaves (reviewed in Samyn-Petit et al, 2003; Arcalis et al, 2013). Glycoproteins that have not passed through the Golgi apparatus typically bear oligomannosidic structures (high-mannose glycans) whereas those in post-Golgi locations tend to bear complex, xylosylated and fucosylated N -glycans.…”

Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective

“…The antibody 2G12 has been expressed in a wide range of plant-based platforms, including wild-type and glycoengineered systems, and has been targeted to different subcellular compartments, thus resulting in a huge variety of glycoforms. The detailed description of these data is beyond the scope of this paper, but overall it was found that the different glycan profiles do not affect the virus-neutralization activity of the antibody in vitro [72]. The impact of the different glycoforms in vivo should be considered in future studies, but it is likely that Fc-mediated antibody effector functions and antibody-dependent cell-mediated cytotoxicity could be influenced by different glycans and this should be considered in the context of systemic antibody administration [70].…”

“…This concept was developed in the EU project Pharma-Planta, which achieved the expression of 2G12 in several plant species and the fast-track development of transgenic tobacco as the primary production platform. The use of many different plants showed that the species, tissue, and subcellular compartment could affect the structure and composition of the antibody glycans, but this had no significant impact on the HIV-neutralization capacity of the antibody in vitro [72]. …”

Glycoengineering in plants for the development of N-glycan structures compatible with biopharmaceuticals

“…3) (Arcalis et al 2013;Drakakaki et al 2006;Fanata et al 2013;Leonard et al 2002;Strasser et al 2007). First, b1,3-GalT produces the Galb1-3Glc-NAc structure by adding b1,3-galactose to non-reducing GlcNAc residues of GlcNAc (1,2) Man 3 XylFucGlcNAc 2 N-glycan (Leonard et al 2002).…”