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.

Keywords: N-acetylglucosaminyltransferase I; Nicotiana benthamiana; glyco-engineered plant; human glucocerebrosidase; plant-made pharmaceuticals.

Figure 1

Generation of GNTI suppression in Nicotiana benthamiana plants. (a) Schematic representation of a construct used to generate GNTI suppression in N. benthamiana plants. 35Sp and 35St represent sequences of the cauliflower mosaic virus 35S promoter and terminator sequences, respectively. Antisense and sense sequences were derived from a coding mRNA sequence of Nicotiana tabacum β‐1,2‐N‐acetylglucosaminyltransferase. (b–c) Protein extracts were separated by 10% SDS‐PAGE and detected by immunoblotting using an antihorseradish peroxidase (anti‐HRP) antibody specific for plant complex‐type N‐glycans. (b) The Nb GNTI‐RNAi transgenic lines 7, 8, 10, 11 and 12 referred to an independent GNTI suppression transgenic line of T ₂ generation N. benthamiana plants. WT, N. benthamiana wild‐type plant; cgl, Arabidopsis thaliana complex‐glycan‐deficient plant. (c) The seven independent transgenic plants of Nb GNTI‐RNAi7 (T ₆ generation) and two independent WT plants (WT1 and WT2) were analysed with anti‐HRP. Silver (S.) staining serves as the loading control.

Figure 2

Glycan profiles of WT and Nb GNTI‐RNAi7 (T ₅ generation). Total N‐glycans from glycoproteins were prepared by hydrazinolysis and labelled with 2‐aminopyridine (PA). PA‐labelled glycans were analysed by RP‐HPLC using a C 18 column. All peaks (indicated by a broken line) were then subjected to LC–MS/MS. The glycan structures identified from deconvoluted MS/MS spectra are indicated in the box. The M5 structure of Nb GNTI‐RNAi7 eluted earlier (indicated by a broken line box) are not included in the calculation.

Figure 3

T ₂ generation of cross‐pollinated Nb GNTI‐RNAi and At‐GC‐HSP19 ( Nb GC ^gnt1 ) Nicotiana benthamiana plants. Total soluble protein (1 μg) was loaded in each lane on 10% SDS‐PAGE gel and then analysed by immunoblotting using (a) anti‐GC antibody or (b) anti‐HRP antibody. (c) Silver staining serves as the loading control.

Figure 4

Glycan characterization of purified GC produced in WT (GC ^WT) and GNTI suppression (GC ^gnt1 ) Nicotiana benthamiana plants. The purified GC ^WT and GC ^gnt1 were analysed on 5%–20% SDS‐PAGE gel and (a) stained with Coomassie Brilliant Blue (1 μg), and also analysed by immunoblot (100 ng) using (b) anti‐GC antibody or (c) anti‐HRP antibody. (a–b) Equal amounts of purified GC ^WT and GC ^gnt1 were analysed while (c) the purified GC ^gnt1 showed a smaller amount of plant complex N‐glycans compared with GC ^WT. (d) One hundred ng of each sample was digested in the absence (−) or presence (+) of either EndoH _f or PNGase F and analysed on 7.5% SDS‐PAGE gel. The GC bands were visualized by immunoblotting.

Figure 5

Nano LC–MS spectra of tryptic glycopeptides derived from purified GC ^WT and GC ^gnt1 . The purified GC ^WT and GC ^gnt1 were excised from a gel, trypsinized and then subjected to nano LC–MS/MS. (a) The elution pattern of tryptic peptide derived from Cerezyme^®, GC ^WT and GC ^gnt1 on nano HPLC. The glycan structures were identified from deconvoluted MS/MS spectra. (b–e) show the glycoforms of glycopeptides with N‐glycosylation sites N19, N59, N146 and N270, respectively.

Figure 6

Uptake of GC ^WT or GC ^gnt1 by macrophages via mannose receptors. The macrophage cells were stained with the macrophage marker CD11b. Unstained cells (negative control, grey) and macrophages (black) were analysed by flow cytometry (a). The effect of mannan on the specific uptake of GC ^WT and GC ^gnt1 was determined (b), and the cellular activities of GC ^WT and GC ^gnt1 were compared (c). The cellular activities of GC ^WT, GC ^gnt1 and Cerezyme^® were compared (d). The results and error bars represent the mean ± SE (n = 3), significantly different at the level of **P < 0.05; *P = 0.05.

Figure 7

Uptake of GC ^WT or GC ^gnt1 into the organs of C57BL/6J mice. The distribution to the liver, spleen, kidney and lungs of mice was evaluated by enzymatic activity at 60 min postinjection. The results and error bars represent the mean ± SE (n = 3 mice).