In Vitro N-Glycan Mannosyl-Phosphorylation of a Therapeutic Enzyme by Using Recombinant Mnn14 Produced from Pichia pastoris.

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

Profiles of plant core-fucosylated N-glycans of acid alpha-glucosidases produced in transgenic rice cell suspension cultures treated with eight different conditions.

Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 µM of 2-fluoro-l-fucose (2-FF), 50 µM of 2-FF, 100 µM of 2-FF, 100 µM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 µM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 µM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.

Production of recombinant human acid ß-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid ß -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

N-glycan Remodeling Using Mannosidase Inhibitors to Increase High-mannose Glycans on Acid α-Glucosidase in Transgenic Rice Cell Cultures.

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Production and Purification of Recombinant Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures.

Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.

Elucidating rice cell metabolism under flooding and drought stresses using flux-based modeling and analysis.

Rice (Oryza sativa) is one of the major food crops in world agriculture, especially in Asia. However, the possibility of subsequent occurrence of flood and drought is a major constraint to its production. Thus, the unique behavior of rice toward flooding and drought stresses has required special attention to understand its metabolic adaptations. However, despite several decades of research investigations, the cellular metabolism of rice remains largely unclear. In this study, in order to elucidate the physiological characteristics in response to such abiotic stresses, we reconstructed what is to our knowledge the first metabolic/regulatory network model of rice, representing two tissue types: germinating seeds and photorespiring leaves. The phenotypic behavior and metabolic states simulated by the model are highly consistent with our suspension culture experiments as well as previous reports. The in silico simulation results of seed-derived rice cells indicated (1) the characteristic metabolic utilization of glycolysis and ethanolic fermentation based on oxygen availability and (2) the efficient sucrose breakdown through sucrose synthase instead of invertase. Similarly, flux analysis on photorespiring leaf cells elucidated the crucial role of plastid-cytosol and mitochondrion-cytosol malate transporters in recycling the ammonia liberated during photorespiration and in exporting the excess redox cofactors, respectively. The model simulations also unraveled the essential role of mitochondrial respiration during drought stress. In the future, the combination of experimental and in silico analyses can serve as a promising approach to understand the complex metabolism of rice and potentially help in identifying engineering targets for improving its productivity as well as enabling stress tolerance.

Process characterization of hCTLA4Ig production in transgenic rice cell cultures using a 3-L bioreactor.

Most of the technical know-how and experience of bioreactor engineering is applicable to plant cell cultures. In this study, transgenic rice cell cultures using RAmy3D promoter were used for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). In process aspect, the rice cells during production phase are strongly influenced by hydrodynamic stresses, such as shear stress and bubble burst. Therefore, the effects of agitation and aeration rates on cell growth and hCTLA4Ig production were investigated in a 3-L multi-bioreactor. By increasing over 240 rpm, the detrimental effects on cell growth and hCTLA4Ig production were observed. At an aeration rate of 0.3 vvm, relative cell viability sharply decreased 2 days earlier than those of lower aeration rates. In addition, it was confirmed that the specific yields and the specific productivity at 0.3 vvm were superior to those values at 0.05 vvm. Overall, higher aeration rate showed the improved hCTLA4Ig production in combination experiment. High aeration rates in general, however, have an undesired effect as excessive aeration was found to negatively affect the quality of hCTLA4Ig. Consequently, the hydrodynamic conditions must be tightly regulated during bioreactor operation in order to enhance hCTLA4Ig productivity and quality in transgenic rice cell cultures.