Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.     

Characterization of the N-linked oligosaccharides of megalin (gp330) from rat kidney

Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.     

Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.     

Determination of nucleotides and sugar nucleotides involved in protein glycosylation by high-performance anion-exchange chromatography: sugar nucleotide contents in cultured insect cells and mammalian cells

We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anion-exchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification. On the other hand, the HPAEC method gave an excellent and reproducible separation of all nucleotides and sugar nucleotides with high sensitivity and reproducibility. We applied the HPAEC method to determine the intracellular sugar nucleotide levels of cultured Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five, BTN-TN-5B1-4) insect cells, and compared them with those in Chinese hamster ovary (CHO-K1) cells. Sf9 and High Five cells showed concentrations of UDP-GlcNAc, UDP-Gal, UDP-Glc, GDP-Fuc, and GDP-Man equal to or higher than those in CHO cells. CMP-Neu5Ac was detected in CHO cells, but it was not detected in Sf9 and High Five cells. In conclusion, the newly developed HPAEC method could provide valuable information necessary for generating sialylated complex-type N-glycans in insect or other cells, either native or genetically manipulated.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.     

Glycan microarrays for screening sialyltransferase specificities

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein-streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human alpha2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcbeta1-4GlcNAc)(n) including N-glycans, rat alpha2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewis(a), human alpha2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1-4 and porcine alpha2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.     

Overexpression of UDP-GlcNAc transporter partially corrects galactosylation defect caused by UDP-Gal transporter mutation

Nucleotide sugar transporters deliver substrates for glycosyltransferases into the endoplasmic reticulum and the Golgi apparatus. We demonstrated that overexpression of UDP-GlcNAc transporter (NGT) in MDCK-RCA(r) and CHO-Lec8 mutant cells defective in UDP-Gal transporter (UGT) restored galactosylation of N-glycans. NGT overexpression resulted in decreased transport of UDP-GlcNAc into the Golgi vesicles. This effect resembled the phenotype of mutant cells corrected by UGT1 overexpression. The transport of UDP-Gal was not significantly changed. Our data suggest that the biological function of UGT and NGT in galactosylation of macromolecules may be coupled.     

Estrogen induced β-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose β-1,4-N-acetylglucosamine (Galβ1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Galβ1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-α-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-α to ERE and that the expressed B4GALT1 plays a crucial role in the proliferation of MCF-7 cells through its activity as a membrane receptor.     

Synthesis of Lewis X epitopes on plant N-glycans

Glycoproteins from tobacco line xFxG1, in which expression of a hybrid β-(1→4)-galactosyltransferase (GalT) and a hybrid α-(1→3)-fucosyltransferase IXa (FUT9a) is combined, contained an abundance of hybrid N-glycans with Lewis X (Le^X) epitopes. A comparison with N-glycan profiles from plants expressing only the hybrid β-(1→4)-galactosyltransferase suggested that the fucosylation of the LacNAc residues in line xFxG1 protected galactosylated N-glycans from endogenous plant β-galactosidase activity.     

Improvement of glycosylation in insect cells with mammalian glycosyltransferases

The N-glycans of recombinant glycoproteins expressed in insect cells mainly contain high mannose or tri-mannose structures, which are truncated forms of the sialylated N-glycans found in mammalian cells. Because asialylated glycoproteins have a shorter half-life in blood circulation, we investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. We co-expressed in two insect cell lines, Sf9 and Ea4, the human alpha1-antitrypsin (halpha1AT) protein with a series of key glycosyltransferases, including GlcNAc transferase II (GnT2), beta1,4-galactosyltransferase (beta14GT), and alpha2,6-sialyltransferase (alpha26ST) by a single recombinant baculovirus. We demonstrated that the enhancement of N-glycosylation is cell type-dependent and is more efficient in Ea4 than Sf9 cells. Glycan analysis indicated that sialylated halpha1AT proteins were produced in Ea4 insect cells expressing the above-mentioned exogenous glycosyltransferases. Therefore, our expression strategy may simplify the production of humanized therapeutic glycoproteins by improving the N-glycosylation pathway in specific insect cells, with an ensemble of exogenous glycosyltransferases in a single recombinant baculovirus.     

Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes

Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51?g/L) and UDP-GlcNTFA (46.54?g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.     

Binding of synthetic sulfated ligands by human splenic galectin 1, a β-galactoside-binding lectin

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.     

Genome-scale identification of UDP-GlcNAc-dependent pathways

Metabolite flux to UDP-GlcNAc and Golgi N-glycan biosynthesis regulates surface residency of glycoprotein receptors and transporters, and thus sensitivities of cells to extracellular cues. Salvage of GlcNAc increases UDP-GlcNAc and branching of N-glycans progressively, but displays an optimum for cell proliferation and bulk endocytosis in mouse NMuMG and human HEK293T epithelial cells. In this report, we measured global changes in gene expression in low and high GlcNAc-supplemented cells. Genes upregulated by high GlcNAc included the EGF and TGF-beta signaling pathways and cell cycle checkpoint, while downregulated genes indicated lower metabolic activity. Genes increased or decreased by high GlcNAc were assessed by transfecting cells with small interfering RNA (siRNA) and measuring effects on three phenotypes: proliferation and bulk endocytosis, and beta1,6GlcNAc-branching of N-glycans. siRNA targeting LGALS3, WBSCR17, PHF3, SDC2 and CTNNAL1 partially reversed the GlcNAc-induced phenotypes, suggesting a role for galectin-3/N-glycans, proteoglycans, O-glycans, and junctional cell adhesion.     

Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN₃ and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.     

Distinct expression profiles of UDP-galactose: β-D-galactoside α1,4-galactosyltransferase and UDP-galactose: β-D-galactoside β1,4-galactosyltransferase in pigeon, ostrich and chicken

We previously identified two novel enzymes in pigeon, α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1-4Gal and Galβ1-4Gal sequences on glycoproteins, respectively. No such glycan structures and/or enzymes have been found in mammals, suggesting that the expression of these enzymes diverged during the course of vertebrate evolution. To compare their expression profiles among avian species, we first established a method for detecting the activities of these two GalTs based on the two-dimensional high pressure liquid chromatography mapping technique, using 2-aminopyridine-derivatized asialo-biantennary N-glycans as an acceptor substrate. When we analyzed the activities of GalTs in pigeon liver extracts in the presence of UDP-Gal, 13 different products containing Galα1-4Galβ1-4GlcNAc, Galβ1-4Galβ1-4GlcNAc and/or Galα1-4Galβ1-4Galβ1-4GlcNAc branches were identified. The newly formed glycosidic linkages of the enzymatic products were determined by nuclear magnetic resonance and methylation analysis, as well as by galactosidase digestions. The activities of both α1,4- and β1,4-GalTs were detected in various tissues in pigeon, although their relative activities were different in each tissue. In contrast, ostrich expressed β1,4-GalT, but not α1,4-GalT, in all tissues analyzed, whereas neither α1,4- nor β1,4-GalT activity was detected in chicken. These results indicate that α1,4- and β1,4-GalTs are expressed in a species-specific manner and are distributed throughout the entire body of pigeon or ostrich when the enzymes are present.     

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.     

The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.