Production of α2,6-sialylated IgG1 in CHO cells.

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and ß1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.

Expression of ß-1,4-galactosyltransferase and suppression of ß-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells.

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of ß-N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of ß-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Characterization of a novel ubiquitin-conjugating enzyme that regulates beta1,4-galactosyltransferase-1 in embryonic stem cells.

In this study we identified a novel galactosyltransferase 1-associating protein (GTAP) by cDNA cloning from a murine embryonic cDNA library using the two-hybrid yeast system. GTAP is expressed in early embryonic tissues, as well as in adult tissues with active cell turnover, and belongs to the class III ubiquitin-conjugating (E2) enzyme family. Its COOH-terminal domain contains a consensus sequence for ubiquitin binding shared by all the ubiquitin-conjugating enzymes, whereas its NH(2)-terminal domain appears critical for the binding and internalization of cell surface galactosyltransferase 1 (GalT1) in embryonic stem cells through a monensin- and MG132-dependent pathway. We have found that GTAP regulates GalT1-associated, laminin-dependent embryonic cell adhesion and the formation of embryoid bodies. Thus, GTAP functions as an evolutionarily conserved E2 enzyme, which may participate in intercellular adhesion and embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Cloning and GST-fused expression in E. coli of mouse beta-1,4-galactosyltransferase.

Beta-1,4-galactosyltransferase (beta4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of beta4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse beta4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopropyl-beta-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse beta4Gal-T. The transcriptional product of beta4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.

Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris is often used as an organism for the heterologous expression of proteins and has been used already for production of a number of glycosyltransferases involved in the biosynthesis of N- and O-linked oligosaccharides. In our recent studies, we have examined the expression in P. pastoris of Arabidopsis thaliana and Drosophila melanogaster core alpha1,3-fucosyltransferases (EC 2.4.1.214), A. thaliana beta1,2-xylosyltransferase (EC 2.4.2.38), bovine beta1,4-galactosyltransferase I (EC 2.4.1.38), D. melanogaster peptide O-xylosyltransferase (EC 2.4.2.26), D. melanogaster and Caenorhabditis elegans beta1,4-galactosyltransferase VII (SQV-3; EC 2.4.1.133) and tomato Lewis-type alpha1,4-fucosyltransferase (EC 2.4.1.65). Temperature, cell density and medium formulation have varying effects on the amount of activity resulting from expression under the control of either the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) or inducible alcohol oxidase (AOX1) promoters. In the case of the A. thaliana xylosyltransferase these effects were most pronounced, since constitutive expression at 16 degrees C resulted in 30-times more activity than inducible expression at 30 degrees C. Also, the exact nature of the constructs had an effect; whereas soluble forms of the A. thaliana xylosyltransferase and fucosyltransferase were active with N-terminal pentahistidine tags (in the former case facilitating purification of the recombinant protein to homogeneity), a C-terminally tagged form of the A. thaliana fucosyltransferase was inactive. In the case of D. melanogaster beta1,4-galactosyltransferase VII, expression with a yeast secretion signal yielded no detectable activity; however, when a full-length form of the enzyme was introduced into P. pastoris, an active secreted form of the protein was produced.

Mammalian glycosyltransferase expression allows sialoglycoprotein production by baculovirus-infected insect cells.

The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.

Efficient preparation of natural and synthetic galactosides with a recombinant beta-1,4-galactosyltransferase-/UDP-4'-gal epimerase fusion protein.

The numerous biological roles of LacNAc-based oligosaccharides have led to an increased demand for these structures for biological studies. In this report, an efficient route for the synthesis of beta-galactosides using a bacterial beta-4-galactosyltransferase/-UDP-4'-gal-epimerase fusion protein is described. The lgtB gene from Neisseria meningitidis and the galE gene from Streptococcus thermophilus were fused and cloned into an expression vector pCW. The fusion protein transfers galactose to a variety of different glucose- and glucosamine-containing acceptors, and utilizes either UDP-galactose or UDP-glucose as donor substrates. A crude lysate from Escherichia coli expressing the fusion protein is demonstrated to be sufficient for the efficient preparation of galactosylated oligosaccharides from inexpensive UDP-glucose in a multigram scale. Lysates containing the fusion protein are also found to be useful in the production of more complex oligosaccharides in coupled reaction mixtures, e.g., in the preparation of sialosides from N-acetylglucosamine. Thus, bacterially expressed fusion protein is well suited for the facile and economic preparation of natural oligosaccharides and synthetic derivatives based on the lactosamine core.

Effect of RQ and pre-seed conditions on biomass and galactosyl transferase production during fed-batch culture of S. cerevisiae BT150.

A feedback RQ controlled fed-batch process for the recombinant production of a soluble human N-deglycosylated recombinant beta-1, 4-galactosyltransferase (NdrGal-T) with Saccharomyces cerevisiae BT150 was investigated. Several RQ values were tested for optimal production of NdrGal-T. Four times higher volumetric activity was reached at RQ=1.0 (32 U l(-1)) than at all higher RQ values (about 8 U l(-1)). RQ, 1.0 was the best choice for both, biomass and enzyme production. Optimal concentration of glucose in preculture was 25 g l(-1). At higher values slightly more ethanol was produced than at lower values of preculture glucose concentrations, moreover no positive effect on biomass and enzyme production was found. Lower values caused not only decrease of ethanol but also decrease of biomass formation (from 1.69 g h(-1) to 0.81 g h(-1)) and enzyme overall productivity (from 2.2 U h(-1) to 0.63 U h(-1)). Successfully performed cultivation with three precultures predicted scale-up possibility of feedback RQ-controlled NdrGal-T production with S. cerevisiae BT150 from lab to pilot-scale fermentor.

The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli.

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

Expression of N-acetyllactosamine and beta1,4-galactosyltransferase (beta4GalT-I) during adenoma-carcinoma sequence in the human colorectum.

We set out to determine the expression profiles of glycoproteins possessing N-acetyllactosamine, a precursor carbohydrate of sialyl Le(x), during colorectal cancer development. We immunohistochemically analyzed the distribution of N-acetyllactosamine as well as of beta4GalT-I, a member of the beta1, 4-galactosyltransferase family responsible for N-acetyllactosamine biosynthesis, in normal mucosa and in adenoma and carcinoma of the human colorectum. Using monoclonal antibody H11, N-acetyllactosamine was barely detectable in the normal mucosa. In low-grade adenoma, however, N-acetyllactosamine was weakly but definitely expressed on the cell surface, and its expression level was moderately increased in high-grade adenoma and markedly increased in carcinoma in situ as well as in advanced carcinoma. To detect beta4GalT-I, we used a newly developed polyclonal antibody (designated A18G), which is specific for the stem region of human beta4GalT-I. Faint expression of beta4GalT-I was detectable in normal mucosa, and the expression level was moderately increased in low-grade adenoma and in high-grade adenoma and markedly increased in carcinoma in situ and advanced carcinoma. The expression of N-acetyllactosamine was highly correlated with the expression of beta4GalT-I in these tumor cells. These results indicate that the expression level of beta4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that beta4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface. (J Histochem Cytochem 47:1593-1601, 1999)

Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns.

beta1,4-Galactosyltransferase (UDP galactose: beta-N-acetylglucosaminide: beta1,4-galactosyltransferase; EC 2.4.1. 22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.

Down-regulation of beta1,4-galactosyltransferase gene expression by cell-cycle suppressor gene p16.

Beta1,4-Galactosyltranferase (beta1,4GT, EC 2.4.1.38) is one of the key enzymes controlling the biosynthesis of complex-type oligosaccharides, and is also one of the best-studied glycosyltransferases. To study the molecular mechanisms involved in the regulation of beta1,4GT gene expression, we transfected cell-cycle suppressor gene p16 into A549 cell line (in which p16 is deleted), measured beta1,4GT gene expression by Northern blot hybridization, and evaluated its activity. It was found that p16 could down-regulate beta1,4GT gene expression and its activity. However, p16 decreased cell surface beta1,4GT activity more than total activity. beta1,4GT mRNA stability was also assayed. It was found that p16 could not influence beta1,4GT mRNA stability.

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.

Agalactosyl IgG and beta-1,4-galactosyltransferase gene expression in rheumatoid arthritis patients and in the arthritis-prone MRL lpr/lpr mouse.

Reduced galactosylation of immunoglobulin G (IgG) is well documented in rheumatoid arthritis (RA), but the reason for this defect is still unknown. There is some evidence supporting a defect in the biosynthetic pathway, and a reduction in the level of beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme activity. Since glycosyltransferases are, in general, regulated at the level of transcription, we have measured the level of beta-1,4-GalTase gene expression in B cells from patients with RA and normal control individuals. We found no significant difference in mRNA levels for the transferase in these two groups (P > 0.7). MRL/Mp-lpr/lpr (MRL-lpr) mice develop a spontaneous arthritis with increased levels of agalactosyl IgG (G0). In spite of a significant reduction in the level of beta-1,4-GalTase mRNA in total spleen lymphocytes from MRL-lpr mice compared with the congenic MRL/Mp-(+/+) (MRL-(+/+) mice and with CBA/Ca mice, we found comparable levels of the beta-1,4-GalTase mRNA in purified B cells from both spleen and lymph nodes of the three strains. Amongst the lymphoid compartments examined, the spleen and peripheral blood were found to be the major contributors of G0 in MRL-lpr mice. These data indicate that in neither human RA, nor in an animal model of this disease, is reduced IgG galactosylation caused by impaired expression of the beta-1,4-GalTase gene in B lymphocytes. Furthermore, splenic B cells, which have normal levels of beta-1,4-GalTase mRNA, appear to be a major source of G0 in MRL-lpr mice.

Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function.

beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.

Golgi localization and in vivo activity of a mammalian glycosyltransferase (human beta1,4-galactosyltransferase) in yeast.

Gene fusions encoding the membrane anchor region of yeast alpha1, 2-mannosyltransferase (Mnt1p) fused to human beta1, 4-galactosyltransferase (Gal-Tf) were constructed and expressed in the yeast Saccharomyces cerevisiae. Fusion proteins containing 82 or only 36 N-terminal residues of Mnt1p were produced and quantitatively N-glycosylated; glycosyl chains were shown to contain alpha1,6-, but not alpha1,3-mannose determinants, a structure typical for an early Golgi compartment. A final Golgi localization of both fusions was confirmed by sucrose gradient fractionations, in which Gal-Tf activity cofractionated with Golgi Mnt1p activity, as well as by immunocytological localization experiments using a monoclonal anti-Gal-Tf antibody. In an in vitro Gal-Tf enzymatic assay the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had comparable Km values for UDP-Gal (about 45 microM). To demonstrate in vivo activity of the Mnt1/Gal-Tf fusion the encoding plasmids were transformed in an alg1 mutant, which at the non-permissive temperature transfers short (GlcNAc)2 glycosyl chains to proteins. Using specific lectins the addition of galactose to several yeast proteins in transformants could be detected. These results demonstrate that Gal-Tf, a mammalian glycosyltransferase, is functional in the molecular environment of the yeast Golgi, indicating conservation between yeast and human cells. The in vivo function of human Gal-Tf indicates that the yeast Golgi is accessible for UDP-Gal and suggests strategies for the construction of yeast strains, in which desired glycoforms of heterologous proteins are produced.

Enhanced galactosyltransferase expression in the failing hearts of spontaneously hypertensive rats.

To examine differences in cardiac gene expression between spontaneously hypertensive rats with and without heart failure, we have used subtractive hybridization to identify differentially expressed genes. After subtraction, cDNAs were amplified by PCR, cloned and sequenced. One of 36 independent cDNAs was found to be 86% homologous to murine UDP-galactose:N-acetylglucosamine beta-1,4-galactosyltransferase. RNA blot analysis confirmed the approximately 4.0 kb rat galactosyltransferase transcript was increased in failing hearts relative to non-failing hearts. Biochemical assay also showed increased galactosyltransferase activity in the failing hearts.

Functional domains of bovine beta-1,4 galactosyltransferase.

A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.