Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage.

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

Recent advances in plant-based bioproduction.

Unable to move on their own, plants have acquired the ability to produce a wide variety of low molecular weight compounds to survive against various stresses. It is estimated that there are as many as one million different kinds. Plants also have the ability to accumulate high levels of proteins. Although plant-based bioproduction has traditionally relied on classical tissue culture methods, the attraction of bioproduction by plants is increasing with the development of omics and bioinformatics and other various technologies, as well as synthetic biology. This review describes the current status and prospects of these plant-based bioproduction from five advanced research topics, (i) de novo production of plant-derived high value terpenoids in engineered yeast, (ii) biotransformation of plant-based materials, (iii) genome editing technology for plant-based bioproduction, (iv) environmental effect of metabolite production in plant factory, and (v) molecular pharming.

The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

Reconstitution of (1→3)-ß-D-glucans measurement system using recombinant Limulus polyphemus Factor G.

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-ß-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and ß from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for ß. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and ß subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and ß subunits for BDG-dependent activation.

Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana.

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine ß1,4-galactosyltransferase (ß1,4-GALT) to modify plant N-glycan with ß1,4-linked Gal residue(s) and Arabidopsis thaliana ß1,3-galactosylatransferase (ß1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine ß1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with ß1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary ß1,4-galactosylated N-glycan structures.

High accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man6-9GlcNAc2 structure (2.8%, 7.1%) and high abundance of the Man5GlcNAc2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man5GlcNAc2 structure, and Man I overexpression enhanced the conversion of Man6-9GlcNAc2 structures to the Man5GlcNAc2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins.

Effects of various disaccharide adaptations on recombinant IgA1 production in CHO-K1 suspension cells.

Immunoglobulin A (IgA) has been showing potential as a new therapeutic antibody. However, recombinant IgA suffers from low yield. Supplementation of the medium is an effective approach to improving the production and quality of recombinant proteins. In this study, we adapted IgA1-producing CHO-K1 suspension cells to a high concentration (150 mM) of different disaccharides, namely sucrose, maltose, lactose, and trehalose, to improve the production and quality of recombinant IgA1. The disaccharide-adapted cell lines had slower cell growth rates, but their cell viability was extended compared to the nonadapted IgA1-producing cell line. Glucose consumption was exhausted in all cell lines except for the maltose-adapted one, which still contained glucose even after the 9th day of culturing. Lactate production was higher among the disaccharide-adapted cell lines. The specific productivity of the maltose-adapted IgA1-producing line was 4.5-fold that of the nonadapted line. In addition, this specific productivity was higher than in previous productions of recombinant IgA1 with a lambda chain. Lastly, secreted IgA1 aggregated in all cell lines, which may have been caused by self-aggregation. This aggregation was also found to begin inside the cells for maltose-adapted cell line. These results suggest that a high concentration of disaccharide-supplemented induced hyperosmolarity in the IgA1-producing CHO-K1 cell lines. In addition, the maltose-adapted CHO-K1 cell line benefited from having an additional source of carbohydrate. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00571-5.

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in Nicotiana benthamiana leaves.

The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium-mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g-1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli-produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts.

Insights into the quality of recombinant proteins produced by two different Bombyx mori expression systems.

The silkworm, Bombyx mori, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been widely used for recombinant protein production in B. mori: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined the quality of the resulting proteins in terms of N-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an N-glycoprotein. Although the production in the baculovirus/silkworm expression system was much more efficient than that in the transgenic silkworm expression system, unexpected variants of IFN-γ were also produced in the former system due to the different N-glycosylation and C-terminal truncations. These results indicate that while high protein production could be achieved in the baculovirus/silkworm expression system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of N-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in B. mori.

Mamestra brassicae NIAS-Mb-32 cell strain 2g2 enables high-yield recombinant protein production in baculovirus-free and baculovirus-based insect cell expression.

The production of recombinant proteins using insect cells has been widely used for over 30 years, which contributing to life science research and biotechnology. Insect cells exhibiting enhanced N-glycosylation and recombinant protein productivity enhance the productivity of the baculovirus-insect cell system (BICS). A new highly proliferative insect cell strain, 2g2, was established from the Mamestra brassicae pupa ovary cell strain NIAS-MB-32 (RCB0413) to address the problem of Sf-rhabdovirus and to explore the newly available possibilities in BICS as well as Sf9, such as increased protein production and recombinant baculovirus amplification. The high-growth cell strain 2g2 was examined for its recombinant protein production ability and baculovirus productivity; moreover, the activity of the produced recombinant proteins was examined using Sf9 as a benchmark. Recombinant protein productivity and virus production by BICS in 2g2 was confirmed as equivalent to that of Sf9. Furthermore, we produced the severe acute respiratory syndrome coronavirus 2 spike protein in a baculovirus-free system and compared its productivity, binding activity with human angiotensin-converting enzyme 2, and N-glycosylation. The productivity and bioactivity were found to be equal to or better than that of Sf9. Moreover, N-glycosylation analysis revealed that the glycans derived from the 2g2-produced glycoproteins were mostly of the high mannose type as Sf9. Therefore, 2g2 may have the same N-glycosylation ability as Sf9. Finally, the Sf-rhabdovirus was confirmed to be negative in 2g2. Our results demonstrated that the novel insect cell strain 2g2 can serve as a protein production tool in scientific research and industrial biotechnology.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

Temporal analysis of N-acetylglucosamine extension of N-glycans in the middle silk gland of silkworm Bombyx mori.

N-glycosylation of proteins is an important post-translational modification in eukaryotic cells. One of the key modifications in protein N-glycosylation is N-acetylglucosamine (GlcNAc) extension mediated by N-acetylglucosaminyltransferase I (GNTI), which triggers N-glycan maturation from high-mannose-type to hybrid- and complex-type structures in Golgi. However, the temporal contributions of GNTI to GlcNAc extension and the resultant N-glycan structures in insects have not been analyzed. Here, focusing on GlcNAc extension of N-glycan in the silkworm Bombyx mori, we analyzed the temporal N-glycan alterations in the middle silk gland (MSG) and characterized the property of key enzyme for complex-type N-glycan biosynthesis, B. mori GNTI (BmGNTI). N-glycan analysis of N-glycoproteins in the MSG demonstrated that BmGNTI identified and characterized in this study consistently contributed to GlcNAc extension of N-glycans, which led to the accumulation of GlcNAc-extended N-glycans as predominant structures throughout the MSG development. The expression profile of GlcNAc extension-related genes revealed that the enzymes contributing to the hydrolysis of GlcNAc showed stage-specific expressions, thereby resulting in accumulations of the end product N-glycans of the enzyme. These results lead to the speculation that not BmGNTI but rather glycosylhydrolases critically influenced the structural formations and the changes in the ratio of N-glycans with GlcNAc residue(s) in MSG.

Effect of fruit maturation on N-glycosylation of plant-derived native and recombinant miraculin.

Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.

Production of recombinant ß-glucocerebrosidase in wild-type and glycoengineered transgenic Nicotiana benthamiana root cultures with different N-glycan profiles.

Gaucher disease is an inherited lysosomal storage disorder caused by an insufficiency of active ß-glucocerebrosidase (GCase). Exogenous recombinant GCase via enzyme replacement therapy is considered the most practical treatment for Gaucher disease. Mannose receptors mediate the efficient uptake of exogenous GCase into macrophages. Thus, terminal mannose residues on N-glycans are essential for the delivery of exogenous GCase. In this study, recombinant GCase was produced in root cultures of wild-type (WT) and glycoengineered transgenic Nicotiana benthamiana with downregulated N-acetylglucosaminyltransferase I expression. Root cultures of WT and glycoengineered transgenic N. benthamiana plants were successfully generated by the induction of plant hormones. Recombinant GCases produced in both root cultures possessed GCase enzyme activity. Purified GCases derived from both root cultures revealed different N-glycan profiles. The WT-derived GCase possessed the predominant plant-type N-glycans, which contain plant-specific sugars-linkages, specifically ß1,2-xylose and α1,3-fucose residues. Notably, the mannosidic-type N-glycans with terminal mannose residues were abundant in the purified GCase derived from glycoengineered N. benthamiana root culture. This research provides a promising plant-based system for the production of recombinant GCase with terminal mannose residues on N-glycans.

Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4,6-dehydratase and GDP-fucose transporter.

Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N-linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00501-3.

Biochemical characterization of Arabidopsis clade F polygalacturonase shows a substrate preference toward oligogalacturonic acids.

Polygalacturonases (PGs) hydrolyze α-1,4-linked d-galacturonic acid (GalUA) in polygalacturonic acid. Previously, PG activity in pea seedlings was found in the Golgi apparatus, where pectin biosynthesis occurs. However, the corresponding genes encoding Golgi-localized PG proteins have never been identified in the higher plants. In this study, we cloned the 5 Arabidopsis genes encoding putative membrane-bound PGs from clade F PGs (AtPGFs) as the first step for the discovery of the Golgi-localized PGs. Five AtPGF proteins (AtPGF3, AtPGF6, AtPGF10, AtPGF14 and AtPGF16) were heterologously produced in Schizosaccharomyces pombe. Among these, only the AtPGF10 protein showed in vitro exo-type PG activity toward fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as a substrate. The optimum PG activity was observed at pH 5.5 and 60°C. The recombinant AtPGF10 protein showed the maximum PG activities toward PA-OGA with 10 degrees of polymerization. The apparent Km values for the PA-OGAs with 7, 11 and 14 degrees of polymerization were 8.0, 22, and 5.9 µM, respectively. This is the first report of the identification and enzymatic characterization of AtPGF10 as PG carrying putative membrane-bound domain.

Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts.

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture.

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Transient Production of Human ß-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme ß-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (ß1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

Bombyx mori ß1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis.

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcß1,2-R with ß1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of ß1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.