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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 GlcNAcβ1,2-R with β1,4-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 β1,2-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.

Insect cells are attractive hosts for biopharmaceutical production due to their high productivity and mammalian-like post-translational modifications. However, their insect-specific N -glycans differ from mammalian types, thereby reducing product desirability. Here, with an emphasis on engineering N -glycosylation in insect cells, we aimed to develop a more tractable Spodoptera frugiperda Sf9 cell platform as a practical alternative to insect-based systems for engineering the production of tri-antennary N -glycans. A database search revealed that silkworm possesses N -acetylglucosaminyltransferase IV (GNTIV), a putative glycosyltransferase essential for tri-antennary N -glycan synthesis; however, it was not functional in vitro. Then, human GNTIV was introduced into insect cells, resulting in the production of small amounts of tri-antennary N -glycans. This suggested the need for additional factors to efficiently biosynthesize tri-antennary N -glycans. Subsequently, additional insect-derived glycosyltransferases, such as active GNTI and/or GNTII, were co-expressed with GNTIV. Co-expression of three N -acetylglucosaminyltransferases effectively led to the increased biosynthesis of tri-antennary N -glycans. On the other hand, trimming of the N -glycan structure was also observed due to the action of one or more endogenous glycosylhydrolases, which hydrolyze the terminal N -acetylglucosamine residue in insect cells. These facts indicate that effective tri-antennary N -glycan biosynthesis in insect cells requires not only the introduction of exogenous glycosyltransferases but also the knockdown or knockout of endogenous glycosylhydrolase(s).

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-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 (β1,2-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.

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 β1,4-galactosyltransferase (β1,4-GALT) to modify plant N -glycan with β1,4-linked Gal residue(s) and Arabidopsis thaliana β1,3-galactosylatransferase (β1,3-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 β1,4-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 β1,3-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 β1,4-galactosylated N -glycan structures.

The mechanism underlying the biosynthesis and accumulation of large quantities of rubber particles and resin in the parenchyma tissue of the stem bark of guayule (Parthenium argentatum Gray) remained unanswered up to now. Here, we focused on rubber particle biosynthesis and accumulation in guayule and performed histochemical analyses using a lipophilic fluorescent dye specific for lipids and spectral confocal laser scanning microscopy. Unmixing images were constructed based on specific spectra of cis-polyisoprene and resin and showed that guayule accumulates a large amount of resin in the resin canals in parenchyma tissue and in pith. Interestingly, the fluorescence signals of rubber were predominantly detected in a specific single layer of epithelial cells around the resin canals. These epithelial cells accumulated large rubber particles and essentially no resin. Immunoblotting and immunostaining of guayule homologue of small rubber particle proteins (GHS), which contributes to the biosynthesis of rubber in guayule, showed that GHS is one of several small rubber particle proteins and is localized around rubber particles in epithelial cells. De novo sequencing of the rubber particle proteins showed the presence of all known organelle proteins, suggesting that epithelial cells biosynthesize rubber particles, followed by remodeling of the cells for the accumulation of rubber particles with subsequent decomposition of the organelles. These results indicate that epithelial cells around resin canals are bifunctional cells dedicated to the biosynthesis and accumulation of rubber particles.

Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-α-L-Rha(1-4)-α-D-GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1–AtRRT4 in liverwort, Marchantia polymorpha , namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the At RRT1 -deficient mutant phenotype in A. thaliana . However, the Mp RRT1 -deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by Mp RRT3 in M. polymorpha and FRB1/ At RRT8 in A. thaliana , the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought.

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 .

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms—EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.Kajiura and Yoshizawa et al. identify three new prenyltransferases in the tree Eucommia ulmoides that synthesize exceptionally high molecular weight trans-1,4-polyisoprene (TPI). Through crystal structure and mutational analyses, they identify key residues required for TPI synthesis and reveal its functional importance in seed development.

Summary 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 β1,2‐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. The plant‐produced lectibody Avaren‐Fc showed enhanced antitumor activity when the core fucose residue was removed from the Fc glycan. Anti‐drug antibodies appeared to augment antitumor efficacy, possibly through the lectibody's vaccinal effect.

Pectin is one of the three key cell wall polysaccharides in land plants and consists of three major structural domains: homogalacturonan, rhamnogalacturonan I (RG-I) and RG-II. Although the glycosyltransferase required for the synthesis of the homogalacturonan and RG-II backbone was identified a decade ago, those for the synthesis of the RG-I backbone, which consists of the repeating disaccharide unit [→2)-α- l -Rha-(1 → 4)-α- d -GalUA-(1→], have remained unknown. Here, we report the identification and characterization of Arabidopsis RG-I:rhamnosyltransferases (RRTs), which transfer the rhamnose residue from UDP-β- l -rhamnose to RG-I oligosaccharides. RRT1, which is one of the four Arabidopsis RRTs, is a single-spanning transmembrane protein, localized to the Golgi apparatus. RRT1 was highly expressed during formation of the seed coat mucilage, which is a specialized cell wall with abundant RG-I. Loss-of-function mutation in RRT1 caused a reduction in the level of RG-I in the seed coat mucilage. The RRTs belong to a novel glycosyltransferase family, now designated GT106. This is a large plant-specific family, and glycosyltransferases in this family seem to have plant-specific roles, such as biosynthesis of plant cell wall polysaccharides. Pectin, the major gelling component of the plant cell wall, is rich in galacturonic acids that compose the backbones of pectic polysaccharides. Now, researchers have identified a new family of enzymes responsible for synthesizing the backbone of pectin, RG-I.