The apiosyltransferase celery UGT94AX1 catalyzes the biosynthesis of the flavone glycoside apiin.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an A. graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physioecological functions of apiose and apiose-containing compounds.

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.

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.

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.

Direct evidence of cytosolic PNGase activity in Arabidopsis thaliana: in vitro assay system for plant cPNGase activity.

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes.

Characterization of Bombyx mori N-acetylglucosaminyltransferase II splicing variants.

N-Acetylglucosaminyltransferase II (GNTII), which catalyzes the transfer of N-acetylglucosamine to N-glycans, plays an essential role in the biosynthesis of branched and complex-type N-glycans. Some characteristics of the GNTIIs from various species have been identified, but not all features have been revealed because some insects have GNTII redundancies due to the possession of splicing variants. In this study, we focused on four splicing variants of silkworm Bombyx mori GNTII (BmGNTII) that differ only in the absence or presence of Exon 2, Exon 9 or both, and we characterized the spatiotemporal transcript levels and enzymatic properties of each. Two of the splicing variants, BmGNTII-B and BmGNTII-D, lack Exon 9, and were expressed more highly in silk glands than any other organs. With respect to the enzymatic properties, optimal temperature and pH were similar among the recombinant BmGNTIIs, but the specific activities and temperature stabilities differed according to the presence or absence of Exon 9 in the splicing variants. These results demonstrate that the B. mori genome encodes splicing variants of GNTII with different enzymatic properties.

Degradation pathway of plant complex-type N-glycans: identification and characterization of a key α1,3-fucosidase from glycoside hydrolase family 29.

Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and ß1,2-linked xylose towards the ß-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex-type N-glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N-glycans. We found that this glycoside hydrolase specifically acted on GlcNAcß1-4(Fucα1-3)GlcNAc, a degradation product of plant complex-type N-glycans, by sequential actions of vacuolar α-mannosidase, ß1,2-xylosidase, and endo-ß-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcß1-4(Fucα1-3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3-fucosidase acting on plant complex-type N-glycans and elucidated the degradation pathway of plant complex-type N-glycans.

Elucidation of rubber biosynthesis and accumulation in the rubber producing shrub, guayule (Parthenium argentatum Gray).

MAIN CONCLUSION: Guayule biosynthesizes and accumulates rubber particles predominantly in epithelial cells in the parenchyma tissue, and this biosynthesis and accumulation is accompanied by remodeling of the roles of epithelial cells. 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.

N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-L-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I.

KEY MESSAGE: Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-L-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man5 (cgl-IDUA) or Man6-8 (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K m) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases.

Fucosyltransferases produce N-glycans containing core l-galactose.

l-Galactose (l-Gal) containing N-glycans and cell wall polysaccharides have been detected in the l-Fuc deficient mur1 mutant of Arabidopsis thaliana. The l-Gal residue is thought to be transferred from GDP-l-Gal, which is a structurally related analog of GDP-l-Fuc, but in vitrol-galactosylation activity has never been detected. In this study, we carried out preparative scale GDP-l-Gal synthesis using recombinant A. thaliana GDP-mannose-3',5'-epimerase. We also demonstrated the l-galactosylation assay of mouse α1,6-fucosyltransferase (MmFUT8) and A. thaliana α1,3-fucosyltransferase (AtFucTA). Both fucosyltransferases showed l-galactosylation activity from GDP-l-Gal to asparagine-linked N-acetyl-ß-d-glucosamine of asialo-agalacto-bi-antennary N-glycan instead of l-fucosylation. In addition, the apparent Km values of MmFUT8 and AtFucTA suggest that l-Fuc was preferentially transferred to N-glycan compared with l-Gal by fucosyltransferases. Our results clearly demonstrate that MmFUT8 and AtFucTA transfer l-Gal residues from GDP-l-Gal and synthesize l-Gal containing N-glycan in vitro.

Altered glycosylation of exported proteins, including surface immune receptors, compromises calcium and downstream signaling responses to microbe-associated molecular patterns in Arabidopsis thaliana.

BACKGROUND: Calcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a "changed calcium elevation" (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin. RESULTS: Here, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles - leading to accumulation of M5(ER), the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor. CONCLUSION: Proper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants.

Recombinant production and characterization of human anti-influenza virus monoclonal antibodies identified from hybridomas fused with human lymphocytes.

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.

Sialylation potentials of the silkworm, Bombyx mori; B. mori possesses an active α2,6-sialyltransferase.

N-Glycosylation is an important post-translational modification in most secreted and membrane-bound proteins in eukaryotic cells. However, the insect N-glycosylation pathway and the potentials contributing to the N-glycan synthesis are still unclear because most of the studies on these subjects have focused on mammals and plants. Here, we identified Bombyx mori sialyltransferase (BmST), which is a Golgi-localized glycosyltransferase and which can modify N-glycans. BmST was ubiquitously expressed in different organs and in various stages of development and localized at the Golgi. Biochemical analysis using Sf9-expressed BmST revealed that BmST encoded α2,6-sialyltransferase and transferred N-acetylneuraminic acid (NeuAc) to the nonreducing terminus of Galß1-R, but exhibited the highest activity toward GalNAcß1,4-GlcNAc-R. Unlike human α2,6-sialyltransferase, BmST required the post-translational modification, especially N-glycosylation, for its full activity. N-Glycoprotein analysis of B. mori fifth instar larvae revealed that high-mannose-type structure was predominant and GlcNAc-linked and fucosylated structures were observed but endogenous galactosyl-, N-acetylgalactosaminyl- and sialyl-N-glycoproteins were undetectable under the standard analytical approach. These results indicate that B. mori genome encodes an α2,6-sialyltransferase, but further investigations of the sialylation potentials are necessary.

Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis.

Asparagine-linked glycans (N-glycans) are crucial signals for protein folding, quality control, and endoplasmic reticulum (ER)-associated degradation (ERAD) in yeast and mammals. Although similar ERAD processes were reported in plants, little is known about their biochemical mechanisms, especially their relationships with N-glycans. Here, we show that a missense mutation in the Arabidopsis EMS-mutagenized bri1 suppressor 3 (EBS3) gene suppresses a dwarf mutant, bri1-9, the phenotypes of which are caused by ER retention and ERAD of a brassinosteroid receptor, BRASSINOSTEROID-INSENSITIVE 1 (BR1). EBS3 encodes the Arabidopsis ortholog of the yeast asparagine-linked glycosylation 9 (ALG9), which catalyzes the ER luminal addition of two terminal α1,2 mannose (Man) residues in assembling the three-branched N-glycan precursor [glucose(Glc)](3)(Man)(9)[N-acetylglucosamine(GlcNAc)](2). Consistent with recent discoveries revealing the importance of the Glc(3)Man(9)GlcNAc(2) C-branch in generating an ERAD signal, the ebs3-1 mutation prevents the Glc(3)Man(9)GlcNAc(2) assembly and inhibits the ERAD of bri1-9. By contrast, overexpression of EBS4 in ebs3-1 bri1-9, which encodes the Arabidopsis ortholog of the yeast ALG12 catalyzing the ER luminal α1,6 Man addition, adds an α1,6 Man to the truncated N-glycan precursor accumulated in ebs3-1 bri1-9, promotes the bri1-9 ERAD, and neutralizes the ebs3-1 suppressor phenotype. Furthermore, a transfer (T)-DNA insertional alg3-T2 mutation, which causes accumulation of an even smaller N-glycan precursor carrying a different exposed α1,6 Man, promotes the ERAD of bri1-9 and enhances its dwarfism. Taken together, our results strongly suggest that the glycan signal to mark an ERAD client in Arabidopsis is likely conserved to be an α1,6 Man-exposed N-glycan.

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.

Transgenic rice seeds accumulating recombinant hypoallergenic birch pollen allergen Bet v 1 generate giant protein bodies.

A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, was previously selected by DNA shuffling of 14 types of Fagales tree pollen allergens. In this study, transgenic rice seed accumulating TPC7 was generated as an oral vaccine against birch pollen allergy by expressing this protein as a secretory protein using the N-terminal signal peptide and the C-terminal KDEL tag under the control of an endosperm-specific glutelin promoter. The highest level of TPC7 accumulation was approximately 207 µg grain(-1). Recombinant TPC7 is a glycoprotein with high mannose-type N-glycan, but without ß1,2-xylose or α1,3-fucose, suggesting that TPC7 is retained in the endoplasmic reticulum (ER). TPC7 is deposited as a novel, giant spherical ER-derived protein body, >20 µm in diameter, which is referred to as the TPC7 body. Removal of the KDEL retention signal or mutation of a cysteine residue resulted in an alteration of TPC7 body morphology, and deletion of the signal peptide prevented the accumulation of TPC7 in rice seeds. Therefore, the novel TPC7 bodies may have formed aggregates within the ER lumen, primarily due to the intrinsic physicochemical properties of the protein.

Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.

UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded N-glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT inhibitory activity are scarce. Here, we report squaryl group-modified UDP analogs as a promising UGGT inhibitor. Among these, the compound possessing a 2'-amino group of the uridine moiety and hydroxyethyl-substituted squaramide exhibited the highest potency, suggesting its relevance as a molecule for further optimization.

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.

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.

Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry ß1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-ß1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.