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

Abstract

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.

Effects of Kifunensine on Production and <em>N</em>-glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

“…due to sugar starvation. The reduction of biomass during sugar starvation was also noticed in our previous reports [30,35] suggesting that kifunensine may not adversely affect rice cell mass cultured in 1.25X NB-S even though a negative impact on rice cell mass cultured in Chu medium without sucrose in the presence of kifunensine was previously reported [25].…”

“…Therefore, to ensure the efficacy of a plant-made biosimilar therapeutic, it is important that the N-glycans are compatible with both the protein's function and the human immune system. There are several strategies for modifying a glycoprotein's N-glycan structures in planta, such as glycoengineering of the host cells using CRISPR/Cas9 genome editing to knock out β(1,2)-xylosyltransferase (XylT) genes and α(1,3)-fucosyltransferase (FucT) genes [10][11][12] and RNA interference (RNAi) technology to down regulate XylT and FucT genes [13][14][15][16][17], targeting of the protein to specific organelles [18][19][20][21], addition of compounds to alter the function of glycan-modifying enzymes [4,[22][23][24][25][26], and in vitro glycan remodeling using chemoenzymatic reaction [27]. In this work, we utilize the last strategy by adding kifunensine, a potent and highly specific inhibitor of mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 (Man8) and Man9GlcNAc2 (Man9) structures [28], to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity.…”

“…In this work, we utilize the last strategy by adding kifunensine, a potent and highly specific inhibitor of mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man8GlcNAc2 (Man8) and Man9GlcNAc2 (Man9) structures [28], to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity. More than a few studies of kifunensine treatment in whole Nicotiana benthamiana plants successfully produced predominant Man9 structure glycoproteins [4,[22][23][24]26]; however, the study of kifunensine treatment in plant cell suspension cultures, including transgenic rice cell suspensions [25], is very limited. In transgenic rice cell suspensions treated with 5 μM kifunensine cultivated in shake flasks, the productivity of a target glycoprotein, acid α-glucosidase (GAA), was significantly lower than the control, but the relative abundance of high mannose structure GAA increased 65% compared to the control [25].…”

“…More than a few studies of kifunensine treatment in whole Nicotiana benthamiana plants successfully produced predominant Man9 structure glycoproteins [4,[22][23][24]26]; however, the study of kifunensine treatment in plant cell suspension cultures, including transgenic rice cell suspensions [25], is very limited. In transgenic rice cell suspensions treated with 5 μM kifunensine cultivated in shake flasks, the productivity of a target glycoprotein, acid α-glucosidase (GAA), was significantly lower than the control, but the relative abundance of high mannose structure GAA increased 65% compared to the control [25]. Here, we report the effects of kifunensine treatment on production and N-glycosylation of a glycoprotein conducted in a bioreactor.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Combined treatment of the mannosidase inhibitors kifunensine (KIF) and swainsonine (SWA) in transgenic rice cell culture media can be an effective method of producing recombinant human acid α-glucosidase (rhGAA) displaying dominantly high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9) glycoforms without genetic manipulation of glycosylation (Choi et al, 2018). In a recent study, knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture was carried out, and it was concluded that the CRISPR/Cas9 system can be utilized for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis (Permyakova et al, 2019).…”