Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.     

Plant cultured cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 [26.8%], GalGNM4 [18.4%], GalGNM3 [19.6%], and GalGNM3X [35.2%]) is different from that found in intracellular glycoproteins (M7A [9.3%], M7B [15.9%], M6B [19.5%], M5 [1.4%], M3X [6.6%], GalGNM5 [35.5%], and GalGNM3 [11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.     

N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin

Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).     

Constitutively active PKB/Akt inhibited apoptosis and down-regulated beta1,4-galactosyltransferase 1 in hepatocarcinoma cells

Beta1,4-galactosyltransferase1 (beta1,4GT1) is localized both in the Golgi complex and on the cell surface. In our previous study, we first reported that beta1,4GT1 was associated with cycloheximide-induced apoptosis in human hepatocarcinoma cells. In this study, we transfected constitutively active protein kinase B (Gag-PKB), a central mediator of anti-apoptotic signals transduced by the PI3-kinase, into SMMC-7721 human hepatocarcinoma cells, and examined its effect on apoptosis and beta1,4GT1 activity. Flow cytometry analysis showed that apoptosis was inhibited in Gag-PKB transfected SMMC-7721 cells. At the same time, beta1,4GT1 mRNA level and enzyme activities were downregulated in these cells, consistent with which, the content of beta1,4 Gal branch in the glycoconjugates was decreased in stably transfected cells. Cotransfection of beta1,4GT1 promoter/luciferase reporter and Gag-PKB decreased the luciferase reporter activity in a dose-dependent manner, indicating that the differences in mRNA levels might be regulated through promoter function. All these findings suggested that changes of beta1,4GT1 activity might be involved in apoptotic pathway in hepatocarcinoma cells.     

Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase- encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N- glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.      

Effect of suppression of TGF-beta1 expression on cell-cycle and gene expression of beta-1,4-galactosyltransferase 1 in human hepatocarcinoma cells

beta-1,4-galactosyltransferase 1 (beta1,4-GT 1) is localized both in the Golgi complex where it catalyzes the transfer of galactose from UDP-galactose to terminal N-acetylglucosamine forming Galbeta1 --> 4GlcNAc structure, and on the cell surface where it serves as an adhesion molecule. It has previously been reported that the expression of beta1,4-GT 1 was cell-cycle-specific, regulated by cell growth. Transforming growth factor-beta1 (TGF-beta1) could regulate cell G1/S phase transition and modulate cell growth in many types of cells. In this study, we introduced the antisense-TGF-beta1 into SMMC-7721 cell, a human hepatocarcinoma cell line, for blocking its intrinsic TGF-beta1 expression, and changing its cell-cycle, and then analyzed the gene expression of beta1,4-GT 1 together with the beta1,4-GT activity. The result showed that the antisense-TGF-beta1 transfected SMMC-7721 cells (AST/7721) were growth enhanced, with more cells in S phase and less cells in G2/M phase compared with the mock transfected cells (pcDNA3/7721). At the same time, it was found that the gene expression of beta1,4-GT 1 in AST/7721 was decreased to one fifth that of pcDNA3/7721, and the cell surface beta1,4-GT activity was reduced to one fifth of the control, while the total activity of beta1,4-GT was decreased to one half that of the control. The results indicate that suppression of TGF-beta1 expression resulted in change of cell-cycle together with the decreased gene expression of beta1,4-GT 1 and beta1,4-GT activity in human hepatocarcinoma cells.     

A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.     

Molecular cloning and expression of a human chondroitin synthase

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.     

Expression of natural human β1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans

β1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human β1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.     

Glyco-engineering of moss lacking plant-specific sugar residues

The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

The widespread effect of beta 1,4-galactosyltransferase on N-glycan processing

We investigated beta 1,4-GalT (UDP-galactose: beta-d-N-acetylglucosaminide beta 1,4-galactosyltransferase) in terms of intracellular competition with GnT-IV (UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1,4-N-acetylglucosaminyltransferase) and GnT-V (UDP-N-acetylglucosamine: alpha1,6-d-mannoside beta 1,6-N-acetylglucosaminyltransferase). The beta 1,4-GalT-I gene was introduced into Chinese hamster ovary (CHO) cells producing human interferon (hIFN)-gamma (IM4/V/IV cells) and five clones expressing various levels of beta 1,4-GalT were isolated. As we previously reported, parental IM4/V/IV cells express high levels of GnT-IVa and -V and produce hIFN-gamma having primarily tetraantennary sugar chains. The branching of sugar chains on hIFN-gamma was suppressed in the beta 1,4-GalT-enhanced clones to a level corresponding to the intracellular activity of beta 1,4-GalT relative to GnTs. Moreover, the contents of hybrid-type and high-mannose-type sugar chains increased in these clones. The results showed that beta 1,4-GalT widely affects N-glycan processing by competing with GnT-IV, GnT-V, and alpha-mannosidase II in cells and also by some other mechanisms that suppress the conversion of high-mannose-type sugar chains to the hybrid type.     

Identification of the full-length coding sequence for human galactosyltransferase (beta-N-acetylglucosaminide: beta 1,4-galactosyltransferase)

A lambda gt11 human placenta cDNA library was screened using a cDNA probe encoding the COOH-terminal region of human beta 1,4-galactosyltransferase and with a synthetic oligonucleotide having a sequence corresponding to that of the 5' end of the cDNA probe. The newly isolated cDNA was found to code for the NH2-terminal and the 5'-untranslated region, primed at an (A)8 region in the coding sequence. A complete amino acid sequence has been deduced which shows only one membrane anchoring domain near the NH2-terminus. Comparison of the sequence to the soluble enzyme suggests proteolytic cleavage at Arg 77. Presently obtained information of human beta 1,4-galactosyltransferase makes it possible to study DNA mutations responsible for genetic defects such as the altered expression of galactosyltransferase found in a variant of congenital dyserythropoietic anemia type II (HEMPAS).     

Involvement of beta-1,4-galactosyltransferase V in malignant transformation-associated changes in glycosylation

In spite of marked changes in the glycosylation upon malignant transformation of cells, no biological significance of beta-1, 4-galactosyltransferase (beta-1,4-GalT) activities has been elucidated. When beta-1,4-GalT activities toward 1 mM GlcNAcbeta-S-pNP were determined using homogenates of NIH3T3 and its transformant, MTAg, MTAg contained 1.3 times higher activities. Northern blot analysis, however, revealed that the beta-1,4-GalT V gene expression increases by three times with a decrease in that of beta-1,4-GalT II by one-fifth and without significant changes in those of other beta-1,4-GalTs in MTAg. Analysis of beta-1,4-GalT V acceptor-specificity showed that the GlcNAcbeta1-->6Man group of the GlcNAcbeta1-->6(GlcNAbeta1-->2)Manalpha1- branch is galactosylated. These results indicate that changes in beta-1,4-GalT II and V activities are important for the altered glycosylation.     

Enhanced activity of recombinant beta-secretase from Drosophila melanogaster S2 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase

beta-Secretase (betaSEC) was expressed in Drososphila melanogaster Schneider 2 (S2) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 56kDa to 61kDa. A lectin blot analysis indicated that recombinant beta-secretase from S2betaSEC/GalT-ST cells (S2 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two dimensional electrophoresis revealed that recombinant beta-secretase from S2betaSEC/GalT-ST cells had a lower isoelectric point compared to beta-secretase from control S2betaSEC cells (S2 cells transformed only with beta-secretase cDNA). Recombinant beta-secretase from transformed S2 cells was also present as heterogeneous forms. The enzyme activity of recombinant beta-secretase from S2betaSEC/GalT-ST cells was enhanced up to 260% compared to control S2betaSEC cells. We have shown that an exogeneous human glycosyltransferases cDNA can be introduced into S2 cells to extend the N-glycan processing capabilities of the insect cell line, and that the extended glycosylation improves the activity of recombinant beta-secretase.     

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.     

Improving solubility of catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino acid replacements.

beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1. The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.