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

Abstract

For the production of therapeutic proteins in plants, the presence of β1,2-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 β1,2-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.

• Authors: Juthamard Limkul, Sayoko Iizuka, Yohei Sato, Ryo Misaki, Takao Ohashi, Toya Ohashi, Kazuhito Fujiyama
• Journal: Plant biotechnology journal (Plant Biotechnol J) Vol. 14 Issue 8 Pg. 1682-94 (08 2016) ISSN: 1467-7652 [Electronic] England
• PMID: 26868756 (Publication Type: Journal Article)
• Copyright: © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Chemical References

• Lectins, C-Type
• Mannose Receptor
• Mannose-Binding Lectins
• Polysaccharides
• Receptors, Cell Surface
• Recombinant Proteins
• N-Acetylglucosaminyltransferases
• alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
• Glucosylceramidase

Topics

• Animals
• Glucosylceramidase (genetics, metabolism, pharmacokinetics)
• Glycosylation
• Humans
• Lectins, C-Type (metabolism)
• Macrophages (drug effects)
• Mannose Receptor
• Mannose-Binding Lectins (metabolism)
• Mice, Inbred C57BL
• N-Acetylglucosaminyltransferases (genetics, metabolism)
• Plants, Genetically Modified
• Pollination
• Polysaccharides (analysis, chemistry, metabolism)
• RNA Interference
• Receptors, Cell Surface (metabolism)
• Recombinant Proteins (genetics, isolation & purification, metabolism)
• Tissue Distribution
• Tobacco (genetics, metabolism)