Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay

Profiling of carbohydrate structures on cell membranes has been difficult to perform because of the complexity and the variations of such structures on cell surface glycans. This study presents a novel method for rapid profiling of cell surface glycans for terminal N-acetyllactosamines (Galβ1-(3)4GlcNAc-R) that are uncapped, capped with sialic acid as SA-Galβ1-(3)4GlcNAc-R, or with α1,3galactosyls as the α-gal epitope- Galα1-3Galβ1-(3)4GlcNAc-R. This method includes two enzymatic reactions: (1) Terminal sialic acid is removed by neuraminidase, and (2) α-gal epitopes are synthesized on the exposed N-acetyllactosamines by α1,3galactosyltransferase. Existing and de novo synthesized α-gal epitopes on cells are quantified by a modification of radioimmunoassay designated as “ELISA inhibition assay,” which measures binding of the monoclonal anti-Gal antibody M86 to α-gal epitopes. This binding is proportional to the number of cell surface α-gal epitopes. The amount of free M86 antibody molecules remaining in the solution is determined by ELISA using synthetic α-gal epitopes linked to albumin as solid phase antigen. The number of α-gal epitopes on cells is estimated by comparing binding curves of M86 incubated with the assayed cells, at various concentrations of the cells, with the binding of M86 to rabbit red cells expressing 2 × 10⁶ α-gal epitopes/cell. We could demonstrate large variations in the number of sialic acid capped N-acetyllactosamines, α-gal epitopes and uncapped N-acetyllactosamines on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species. This method may be useful for rapid identification of changes in glycosylation patterns in cells subjected to various treatments, or in various states of differentiation.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Production of sialyltrisaccharides using β-galactosidase andtrans-sialidase in one pot

Sialyltrisaccharides based on β-galactosyldisaccharides were synthesized using β-galactosidase andtrans-sialidase in one pot. Using β-galactosidase fromBacillus circulans andtrans-sialidase fromTrypanosoma cruzi simultaneously, 6 mM sialyltrisaccharides composed of about 95% NeuAcα(2,3)Galβ(1,4)GlcNAc and 5% NeuAcα(2,3)Galβ(1,6)GlcNAc were produced from a reaction mixture containing 25 mM 0-nitrophenyl-β-D-galactopyranoside, 100 mM N-acety lglucosamine and 10 mM p-nitrophenyl-α-D-N-acetylneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylglucosamine was prevented. Using β-galactosidase fromEscherichia coli and the sametrans-sialidase, 15 mM sialyltrisaccharides composed of about 90% NeuAcα(2,3)Galβ(1,6)GlcNAc and 10% NeuAcα(2,3)Galβ (1,4)GlcNAc were produced from a reaction mixture containing 400 mM galactose, 800 mM N-acetylglucosamine and 20 mMp-nitrophenyl-α-D-N-acetylneuraminic acid. In this study, the reverse-galactosylation reaction between galactose and N-acetylglucosamine was dominant since the disaccharide intermediate mainly resulted in the sialylated product.     

Brief report: a new profile of terminal <Emphasis Type="Italic">N</Emphasis>-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galβ1-4GlcNAc-R) and α-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galβ1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack α-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that α-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-α-Gal epitopes. Whereas, the expression of the α-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

Syntheses of mucin-type <Emphasis Type="Italic">O</Emphasis>-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of <Emphasis Type="Italic">Bifidobacterium</Emphasis> endo-α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.     

Sprouting of the fructan- and starch-storing geophyte Lachenalia minima: Effects on carbohydrate and water content within the bulbs

N-linked glycans of wall-bound exo- β -glucanases from mung bean and barley seedlings, namely Mung-ExoI and Barley-ExoII, were characterized. The N-linked glycans of Mung-ExoI and Barley-ExoII were liberated by gas-phase hydrazinolysis followed by re-N-acetylation. Their structures were determined by two-dimensional sugar-mapping analysis and MALDI-TOF mass spectrometry. N-glycans from both glucanases were of paucimannosidic-type (small complex-type) structures, Man α 1-6(±Man α 1-3)(Xyl β 1-2)Man β 1-4GlcNAc β 1-4(±Fuc α 1-3) GlcNAc, which are known as typical vacuole-type N-glycans. The results suggest that N-glycans of cell-wall glucanase were produced by partial trimming of complex-type N-glycans by exoglycosidases during its transport from Golgi apparatus to cell walls or in the cell walls.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

Specificity analysis of the C-type lectin from rattlesnake venom,and its selectivity towards Gal- or GalNAc-terminated glycoproteins

The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans, terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a K_D for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with a ten-fold better K_D for GalNAc.     

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with β1–4, α1–6 and β1–6 glycosidic bond were synthesized; Galβ1–4GlcNH₂, Galα1–6GlcNH₂, Galα1–6GlcNAc, Galβ1–6GlcNH₂, Galβ1–4Galβ1–4GlcNH₂ and Galβ1–4Galβ1–4GlcNAc. Galα1–6GlcNH₂ (MelNH₂) and glucosamine (GlcNH₂) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH₂. Adding Galα1–6GlcNH₂ or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galα1–6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galα1–6GlcNH₂ were stained. The difference in the stainability of the K562 cells by Galα1–6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galα1–6GlcNH₂ and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galα1–6GlcNH₂ binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (γ) antibodies).     

Structure determination of a sulfated N-glycans, candidate for a precursor of the selectin ligand in bovine lung

To clarify the structure of non-sialic acid anionic residue on N-glycans in the mammalian tissues, we have isolated sialidase-resistant anionic residue on N-glycans from bovine lung. Analyses by partial acid hydrolysis and glycosidase digestions combined with a two-dimensional HPLC mapping method revealed that the major sialidase-resistant anionic N-glycan had a fucosylbianntenary core structure. The anionic residue was identified as a sulfate ester by methanolysis, anion-exchange chromatography, and mass spectrometry. The linkage position of the sulfate ester was the 6-position of the GlcNAc residue on the Manα1-6 branch. This conclusion was based on the results of glycosidase digestions followed by two-dimensional HPLC mapping. Furthermore, the disialylated form of this sulfated glycan was dominant, and no asialo form was detected. The structure of the major anionic N-glycan prepared from bovine lung and having a sulfate was proposed to be the pyridylamino derivative of Siaα2-3Gαlβ1-4(HSO₃-6)GlcNAcβ1-2Manα1-6(Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-³H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glycomic studies of Drosophila melanogaster embryos

With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galβ1-3GalNAc sequence.     

Glycan-binding profile of DC-like cells

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.     

Structural and immunochemical identification of Lea, Leb, H type 1, and related glycolipids in small intestinal mucosa of a group O Le(a-b-) nonsecretor

Total nonacid glycosphingolipids were isolated from small intestine mucosal scrapings of a red cell blood group O Le(a-b-) nonsecretor cadaver. Glycolipids were extracted and fractionated into five fractions based on chromatographic and immunostaining properties. These glycolipid fractions were then analysed by thin-layer chromatography for Lewis activity with antibodies reactive to the type 1 precursor (Lec), H type 1 (Led), Lea and Leb epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and EI-MS/MS-TOF) and proton NMR spectroscopy. EI-MS/MS-TOF allowed for the identification of trace substances in fractions containing several other glycolipid species. Consistent with the red cell phenotype, large amounts of lactotetraosylceramide (Lec-4) were detected. Inconsistent with the red cell phenotype, small quantities of Lea-5, H-5-1 and Leb-6 glycolipids were immunochemically and structurally identified in the small intestine of this individual. By EI-MS/MS-TOF several large glycolipids with 9 and 10 sugar residues were also identified. The extensive carbohydrate chain elongation seen in this individual with a Lewis negative nonsecretor phenotype supports the concept that Lewis and Secretor blood group fucosylation may be a mechanism to control type 1 glycoconjugate chain extension. Abbreviations: FUT1, H gene; FUT2, Secretor gene, (gene bank accession no. U17894); FUT3, Lewis gene or Fuc-TIII gene, (gene bank accession no. X53578); FUT5, Fuc-TV gene; [Imm]+, immonium ion; Lea-5, Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Leb-6, Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lec-4, Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Led or H-5-1, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Lex-5, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; MAb, monoclonal antibody; MS, mass spectrometry; CID, collision-induced dissociation; EI, electron impact ionisation; MS/MS-TOF, tandem mass spectrometry using a time-of-flight mass spectrometer as the second mass spectrometer: m/Cz, mass-to-charge ratio; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; TLC, (high performance) thin layer chromatography. Saccharide types are abbreviated to Hex for hexose, HexNAc for N-acetylhexosamine and dHex for deoxyhexose (fucose). Ceramide is abbreviated to Cer, and ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Convenient and rapid analysis of linkage isomers of fucose-containing oligosaccharides by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSⁿ spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.     

Perspectives on the significance of altered glycosylation of glycoproteins in cancer

No abstract Abbreviations: Sia, sialic acid, type unspecified; Tn antigen, GalNAcα 1-O-Ser/Thr; T antigen, Galβ1-3GalNAcα-O-Ser/Thr; Sialyl LewisX, Siaα2-3Galβ1-4(Fucα1-3)GlcNAc; Sialyl Lewisa, Siaα2-3Galβ1-3(Fucα1-4)GlcNAc; Sialyl-Tn antigen, Siaα2-6GalNAcα1-O-Ser/Thr; FucT, fucosyltransferase; ST, sialyltransferase. This revised version was published online in November 2006 with corrections to the Cover Date.