Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

Abstract

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)−1 or 7.5 ± 0.4 mg L−1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.

Electrophysiological evaluation of an anticancer drug gemcitabine on cardiotoxicity revealing down-regulation and modification of the activation gating properties in the human rapid delayed rectifier potassium channel

“…To further characterize the effect of gemcitabine on the hERG channel glycosylation disruption, a specific class I mannosidase inhibitor kifunensine [ 28 ] was applied in combined with gemcitabine. Fig 8A demonstrates representative I hERG in vehicle (48 h), in kifunensine alone (48 h), and in kifunensine (48 h) combined with gemcitabine treatment (24 h).…”

Disrupting N-Glycosylation Using Type I Mannosidase Inhibitors Alters B-Cell Receptor Signaling

“…However, considerations of relevant cell types are important depending on the applications of the studies as the biological effects of Man I inhibition can vary depending on the type of cells used. The acylated derivatives of kifunensine we used in this study were functionally equivalent to kifunensine, but their higher potency, mediated by their higher hydrophobicity, may help overcome the mass-transfer limitation of kifunensine. ,− Therefore, these kifunensine derivatives will be useful in supporting research investigating the therapeutic potential of inhibiting Man I, as well as cost-effective alternatives for generating high mannose glycoprotein therapeutics …”

“…Specifically, it has been shown to inhibit endoplasmic reticulum and Golgi localized Man I enzymes . Therefore, kifunensine mediated disruption of the N-glycosylation biosynthetic pathway results in an increased prevalence of high-mannose N-glycans and a reduction in the formation of hybrid- and complex-type N-glycans. , Kifunensine has also been reported to have therapeutic potential for the treatment of ERAD-related diseases , (e.g., Alzheimer’s disease, lysosomal storage disorders, catecholaminergic polymorphic ventricular tachycardia, sarcoglycanopathies , ), cancer, − viral infections, − and it is also applied for the preparation of high-mannose N-glycan bearing glycoprotein therapeutics. ,,− However, the widespread use of kifunensine is limited by its high hydrophilicity, which hampers its ability to permeate tissue and enter cells (i.e., mass-transfer limitations). ,− We have previously described the preparation of acylated derivatives of kifunensine (i.e., JDW-II-004 and JDW-II-010) that exhibit >75-fold greater potency than kifunensine in assays leveraging immortalized cells …”

Novel Proteome and N-Glycoproteome of the Thermophilic Fungus Chaetomium thermophilum in Response to High Temperature

“…In the differentially modified GO enrichment-based clustering analysis, unfolded protein binding and glycosylation-related proteins were upregulated enrichment in T55/T50, and carbohydrate metabolic processes are enriched in T50/T30 and T55/T30, indicating that N-glycosylation could affect carbohydrate metabolic processes and protein folding. N-linked glycans could provide blueprints to precisely instruct the folding of protein substrates ( Xu and Ng, 2015 ; Jayaprakash and Surolia, 2017 ; Macharoen et al, 2020 ). In KEGG pathway analyses, the mRNA surveillance pathway and protein processing in the endoplasmic reticulum are upregulated enrichment, autophagy, and cell cycle, and varying types of N-glycan biosynthesis were downregulated enrichment.…”