Analysis of N-glycan profile of Arabidopsis alg3 cell culture.

N-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man3GlcNAc2 (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N-glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy.

The production of human glucocerebrosidase in glyco-engineered Nicotiana benthamiana plants.

For the production of therapeutic proteins in plants, the presence of ß1,2-xylose and core α1,3-fucose on plants' N-glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down-regulate the endogenous N-acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco-engineered line (NbGNTI-RNAi) showed a strong reduction of plant-specific N-glycans, with the result that as much as 90.9% of the total N-glycans were of high-mannose type. Therefore, this NbGNTI-RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI-RNAi plant was cross-pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N-glycan structures that were presented on all of the four occupied N-glycosylation sites of recombinant GC in NbGNTI-RNAi plants (GC(gnt1) ) showed that the majority (ranging from 73.3% up to 85.5%) of the N-glycans had mannose-type structures lacking potential immunogenic ß1,2-xylose and α1,3-fucose epitopes. Moreover, GC(gnt1) could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI-RNAi line, producing GC, was stable and the NbGNTI-RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N-glycan structures.

The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.