GlcNAc Beta Research Articles

In vitro biosynthesis of a decasaccharide prototype of multiply branched polylactosaminoglycan backbones.

Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.

Structural analysis of the oligosaccharide-alditols released by reductive beta-elimination from the jelly coat of Rana utricularia eggs.

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Rana utricularia were analysed by 1H-NMR spectroscopy. Comparison of their structures with those characterized from seven other amphibians confirms that the carbohydrate chains of the jelly coat mucins are markers of the species. The new sequence GlcNAc(beta1-3)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc-ol is characteristic of Rana utricularia. The presence of blood group A determinants constitutes the main feature of this mucin.

Prenatal alcohol exposure affects galactosyltransferase activity and glycoconjugates in the Golgi apparatus of fetal rat hepatocytes.

Prenatal exposure to alcohol affects the morphological, structural, and functional features of the Golgi apparatus (GA), thus altering the glycosylation process in fetal hepatocytes. To elucidate the cellular mechanisms underlying these alterations, we have studied the effect of alcohol exposure in utero on the activity of liver galactosyltransferase, an enzyme involved in the glycosylation process, and on the hepatic glycoprotein sugar composition. For this, livers from 21-day-old fetuses obtained from control and ethanol-fed rats were used. Galactosyltransferase (GT) activity was determined in isolated GA cis and trans fractions. Colloidal gold-labeled lectin cytochemistry was used to analyze sugar residues in hepatocytes at the subcellular level. Finally, the integrity of the GA after alcohol treatment was assessed by electron microscopy and by evaluating the distribution of the Golgi beta-COP, a protein involved in vesicular trafficking. Prenatal alcohol exposure induces a significant increase in both liver weight and total protein content in the trans Golgi. Moreover, this treatment decreases the activity of galactosyltransferase, increases alpha-L-Fuc residues, and reduces the number of alpha-Man, GlcNAc(beta1,4,GlcNAc)1,2, GalNAc alpha1,3GalNAc, alpha-GalNAc, and a-Gal residues. Alcohol exposure also causes the Golgi cisternae to disappear in about 30% of the hepatocytes, and reduces 75% the number of anti-Golgi beta-COP protein binding sites. Our results suggest that the decrease in galactosyltransferase activity, the alterations in the oligosaccharide chain composition, and the reduction in the amount of Golgi beta-COP protein could be involved in the alterations in the glycosylation process, as well as in the accumulation of hepatic proteins observed after prenatal alcohol exposure. These alterations could contribute, therefore, to the alcohol-induced injury in the developing liver.

Proton NMR study of triantennary complex type N-linked glycan chains: assignment of proton chemical shifts of the beta-Man residue in a basic unit of the triantennary glycan chain having a GlcNAc beta 1-->6 Man alpha 1-->6 Man beta-->sequence.

The chemical shifts of ring protons of the beta-Man residue in a triantennary complex type N-linked glycan chain having a GlcNAc beta 1-->6(GlcNAc beta 1-->2)Man alpha 1-->6 Man beta sequence were unambiguously determined by two-dimensional proton nuclear magnetic resonance (1H-NMR) spectroscopic methods. The chemical shift of H4 (3.84 ppm) of the beta-Man residue was for the first time revealed to be different from those (approximately 3.77 ppm) of biantennary and alternative type of triantennary glycans having a GlcNAc beta 1-->2 Man alpha 1-->6 Man beta sequence, but quite close to that (3.86 ppm) of a pentaantennary glycan containing a GlcNAc beta 1-->6 residue on the Man alpha 1-->6 Man beta sequence. Thus, the addition of GlcNAc beta 1-->6 residue on the Man-4' residue, whose formation is catalyzed by GlcNAc transferase V, is considered to cause a down-field shift of beta-Man H4 in the complex-type N-glycan chains. One possible explanation of this phenomenon is that the conformation of Man alpha 1-->6 arm is folded back toward the proximal core region, as is the case with the complex-type N-glycan chains with the bisecting GlcNAc residue.

Synthesis of a new nanomolar saccharide inhibitor of lymphocyte adhesion: different polylactosamine backbones present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode.

Lymphocyte infiltration is a hallmark of acute rejections in solid organ transplants, such as cardiac allograft. We have previously shown that lymphocyte extravasation to cardiac grafts undergoing rejection is largely due to interactions between lymphocyte L-selectin and its sialyl Lewis x (sLex) decorated ligands. Our previous work demonstrated further that an enzymatically synthetized tetravalent sLex glycan of a branched polylactosamine backbone is a highly efficient inhibitor of L-selectin-dependent lymphocyte adhesion to graft endothelium. To improve the availability of multivalent sLex glycans for anti-inflammatory indications, we now report enzymatic synthesis of another tetravalent sLex glycan that can be potentially produced on a large scale, and show that even the new saccharide is a nanomolar inhibitor of L-selectin-dependent lymphocyte adhesion. The novel antagonist is sLex beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc (8) (where LacNAc is the disaccharide Gal beta 1-4GlcNac and sLex is the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc). Its five-step synthesis was started from the octameric polylactosamine LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc (3), which in turn is accessible in one step from the hexasaccharide LacNAc beta 1-3'LacNAc beta 1-3'LacNAc. Importantly, the hexasaccharide primer has been synthesized chemically (Alais and Veyrieres, Tetrahedron Lett., 24, 5223, 1983). Hence, our data outline a route to glycan 8, consisting of a combination of chemical and enzymatic methods of oligosaccharide synthesis. In addition, our data show that polylactosamine backbones are able to present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode, without a requirement for uniqueness in the backbone structure.

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda:Ascaridida).

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(beta 1-1)ceramide, Man(beta 1-4)Glc(beta 1-1)ceramide, GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)ceramide and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine.

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type.

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.

Purification and partial characterization of beta-galactosidase from Tritrichomonas foetus.

The work presented in this paper describes the purification and properties of a beta-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange and affinity chromatography resulted in a high level of purification. After purification beta-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The beta-galactosidase isolated had a pH optimum of 5.8. The Km determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-beta-glucosaminidase, alpha-galactosidase and N-acetyl-alpha-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads are known to be apathogenic in semen. The purified beta-galactosidase was found to have the capacity to hydrolyse lactose (Gal beta1-4 Glc), lacto-N-biose 1 (Gal beta1-3 GlcNAc) and N-acetyllactosamine (Gal beta1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl beta-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl beta-glucopyranoside a band was detected which did not coincide with that of beta-galactosidase. Since the beta-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the beta-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude to cloning and further study of the gene.

Characterization of lactoferrin-binding proteins of human macrophage membrane: multiple species of lactoferrin-binding proteins with polylactosamine-binding ability.

Human lactoferrin (LF) specifically binds to human monocytic leukemia cell line THP-1 cells differentiated into macrophages, and it has been suggested that the poly-N-acetyllactosaminyl saccharide chains of LF are involved. We partially purified and characterized LF-binding proteins with affinity for polylactosamines from THP-1 cells. LF-binding activity was solubilized by nonionic detergent Triton X-100 from THP-1 cell membrane, and subjected to affinity chromatography using an LF-Sepharose column. LF-binding activity, detected by ligand blotting assay, was eluted and further fractionated by affinity chromatography using a Sepharose column coupled with band 3, a polylactosaminyl chain-containing glycoprotein of human erythrocyte membrane. LF-binding activity was separated into three fractions (frs. B1, B2, and B3). These fractions exhibited band 3-binding activity as detected by ligand blotting assay. Dodecylsulfate-polyacrylamide gel electrophoresis of frs. B1, B2, and B3, followed by detection of LF-binding activity on Western blots, indicated that frs. B1, B2, and B3 contained LF-binding proteins with a molecular mass of 35, 50 and 80, and 35-37 kDa, respectively. Binding of LF to each of the fractions on the dot blots was partially inhibited by LF oligosaccharides, band 3 oligosaccharides and lacto-N-neotetraose, each containing di-N-acetyllactosaminyl or analogous structure, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc (or Glc). These results suggest that the 35, 50 and/or 80, and 35-37 kDa proteins on THP-1 cells are LF-binding proteins with polylactosamine-binding ability.

Enzymatic syntheses of GlcNAc beta 1-2Man and Gal beta 1-4GlcNAc beta 1-2Man as components of complex type sugar chains.

GlcNAc beta 1-2Man and GlcNAc beta 1-6Man were synthesized using the reverse hydrolysis activity of beta-N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal beta 1-4GlcNAc beta 1-2Man and Gal beta 1-4GlcNAc beta 1-6Man were synthesized regioselectively using the transglycosylation activity of beta-galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies.

Rodents infected with Schistosoma mansoni produce cytolytic IgG and IgM antibodies to the Lewis x antigen.

Schistosoma mansoni is a blood fluke that produces glycoconjugates containing the Lewis x antigen (Le(x)) Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->R. However, Le(x) antigen is also normally expressed in many tissues of adult rodents. We now report that mice and hamsters chronically infected with S.mansoni generate high titers of both IgM and IgG antibodies reactive with Le(x) and that no reactivity is present in sera from uninfected animals. Anti-Le(x) antibodies were detected by ELISA using the Le(x)-containing neoglycoprotein lacto-N-fucopentaoseIII-BSA. The IgG in infected animals consists of IgG1, IgG2a, and IgG2b subclasses and binds to Protein A-Sepharose. The sera of infected animals reacts only with Le(x) antigen and has no reactivity toward either Le(a) or sialyl Le(x). The IgM response to Le(x) is detectable at week 2, whereas the IgG response is detectable at weeks 5-6 following infection of mice. The sera of infected mice and hamsters can mediate the complement-dependent cytolysis (CDC) of cells expressing surface Le(x). This cytolytic activity is exclusively effected by the anti-Le(x) antibodies, since their removal from sera by adsorption depletes the sera of CDC activity. Thus, the abundant expression of the Le(x) antigens by the parasite elicits cytolytic antibodies reactive with a host antigen.

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.

A defined set of oligosaccharides and glycopeptides containing alpha-linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Le(x)) Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc. The lectin did not bind glycans containing either sialylLe(x) or VIM-2 determinants, nor did it bind the isomeric Le(x), Gal beta 1-3[Fuc alpha 1-4]GlcNAc-R. Although 2'-fucosyllactose Fuc alpha 1-2Gal beta 1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc alpha 1-2Gal beta 1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Le(x) antigen and is useful in analyzing specific fucosylation of glycoconjugates.

The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells.

A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta1,2) Man(alpha1,6) Man(beta1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas-liquid chromatography-mass spectrometry.

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

A new monoclonal antibody (TU-1) directed against the Gal alpha 1-4Gal beta 1-4Glc 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(Gal alpha 1-4Gal beta 1-4Glc beta 1-1'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 (Gal beta 1-4Glc beta 1-1'Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer), galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), sulfatide (HSO3-3Gal beta 1-1'Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Gal alpha 1-4Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'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.

A monoclonal antibody directed against high Mr salivary mucins recognizes the SO3-3Galβ1-3GlcNAc moiety of sulfo-Lewisa: a histochemical survey of human and rat tissue

Using a panel of synthetic oligosaccharides attached to a polyacrylamide carrier, the epitope of monoclonal antibody F2, evoked to high M(r) salivary mucins, was mapped to the SO3-3Gal beta 1-3GlcNAc- moeity of the sulfo-Le(a) antigen. Using immunochemical techniques, the expression of the F2-epitope was investigated in a number of different isolated human mucin species, as well as in human and rat tissue specimens. The mAb F2 bound to high M(r) salivary mucins, cervical mucins, colon mucins and gallblader mucins, but not to low M(r) salivary mucins nor to gastric mucins. Immunohistochemical screening of human tissues with mAb F2 revealed a positive reaction with a number of epithelia, including the (sero)mucous salivary glands, the goblet cells of the colon, submucosal glands of the lung, the lining epithelium of cervical and esophageal glands, the suprabasal skin keratinocytes, and Hassall's corpuscles of the thymus. No staining was found in normal breast, pancreas, small intestine, spleen, and lymph nodes. Normal gastric glands were negative, but gastric intestinal metaplastic glands strongly stained with the antibody. In rat tissues, mAb F2 labeled epithelial cells of salivary glands, colon and stomach. In addition to epithelial cells, extracellular matrix components in rat thymus and skin were labeled by mAb F2. No labeling of erythrocytes, granulocytes, lymphocytes or bone marrow cells was found by FACScan analysis. The present data shows a tissue specific distribution of the F2-epitope in cells from the epithelial lineage in human and rat.

A novel glycosphingolipid expressed in pig kidney: Gal alpha 1-3Lewis(x) hexaglycosylceramide.

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Gal alpha 1-3Gal determinant revealed several glycosphingolipids reacting with different intensities. A minor glycosphingolipid was isolated by preparative high performance thin layer chromatography. It was characterized as a type 2 hexaglycosylceramide with the following structure Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with alpha-galactosidase followed by immunostaining with an anti-Lewis(x) monoclonal antibody. The proton NMR spectrum was found compatible with the proposed structure. Two other glycosphingolipids carrying the new determinant were partially characterized as an octa- and a branched-dodecaglycosylceramide. The expression of the Gal alpha 1-3 Lewis(x) determinant appeared to be developmentally regulated as it increased with age. The characterization of Gal alpha 1-3Le(x) in pig kidney indicates a new epitope capable of recognition by human natural antibodies in the context of xenotransplantation of pig organs to man. It also adds new members to the family of Le(x)-based glycolipids.

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Multiple Non-reducing Chain Termini Isolated from Bovine Corneal Keratan Sulfates

Keratan sulfate-containing proteoglycans were isolated from bovine cornea (15-month-old to 3-year-old animals) and digested with the enzyme, keratanase II. The released oligosaccharides, which included non-reducing termini and repeat region oligosaccharides but not linkage regions, were reduced with alkaline borohydride and fractionated on a Spherisorb column. These oligosaccharides were examined by 600-MHz 1H NMR spectroscopy using one- and two-dimensional methods and, in addition to some oligosaccharide alditols previously recovered from skeletal keratan sulfate, the following new capping structures were identified: NeuAcalpha2-6Galbeta1-4GlcNAc(S)-ol, NeuAcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-6Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4Gl cNA c(S)-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc(S )-ol, NeuGcalpha2-3Gal(S)beta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-o l, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Galbeta1-4GlcNAc( S)-ol, Galalpha1-3Galbeta1-4GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)- ol, GlcNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol, and GalNAc(S)beta1-3Gal(S)beta1-4GlcNAc(S)-ol. These structures represent seven families of capping residues, whose relative molar proportions are given in parentheses: NeuAcalpha(2-3)- (12%), NeuAcalpha(2-6)- (41%), NeuGcalpha(2-3)- and NeuGcalpha(2-6)- families (12%), Galalpha(1-3)- (26%), GalNAc(S)beta(1-3)- (5%), and GlcNAc(S)beta(1-3)- (4%). It is not clear, at present, where each of these structures occurs on the bi-antennary N-linked corneal keratan sulfate chains, which themselves occur within three keratan sulfate proteoglycan species. However, examination of the relative proportions of the capping to the repeat structures and knowledge of the average molecular size suggests that the sum of these non-reducing termini represents the caps of two antennae.

Expression of Phaseolus vulgaris leukoagglutinin-binding oligosaccharides in oral squamous cell carcinoma: possible association with the metastatic potential.

The expression of -GlcNAc beta 1-6Man-(beta 1-6) branched oligosaccharides in carcinoma cells has been considered to influence their metastatic potentials. In the present paper, the lectin histochemistry of oral squamous cell carcinomas obtained in biopsy from 34 patients with Phaseolus vulgaris leukoagglutinin (L-PHA), which potentially binds to N-glycosidic carbohydrates with beta 1-6 linked lactosamin antennae, was studied in order to analyze the relationship between their staining patterns and metastases. The L-PHA-binding oligosaccharides of the carcinomas were expressed on the cell surface in the following patterns: (i) all cells were positive for the staining ('positive'); (ii) some cells were positive but the rest of the carcinoma cells were negative ('weakly positive'); and (iii) all were negative ('negative'). Statistical analysis revealed that the incidence of the metastasis to regional lymph nodes in the 'positive' cases was significantly higher than that in the 'negative' cases. Moreover, the number of the CD14 positive cells including macrophages in the stroma adjacent to the carcinomas in the 'positive' cases was less than that in the 'negative' or 'weakly positive' cases. The expression of L-PHA-binding oligosaccharides in oral squamous cell carcinoma may be responsible for their metastatic potential to regional lymph nodes, possibly including their ability to escape macrophage recognition.

One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration.

The trisaccharide Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was enzymatically synthesized, with in situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4'-epimerase, UDP-Gal:GlcNAc beta 4-galactosyltransferase and UDP-Gal:Gal beta 1-->4GlcNAc alpha 3-galactosyltransferase, Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was formed in a 2.2 mumol ml-1 yield starting from the acceptor GlcNAc beta 1-->O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc epitope which pays an important role in various biological and immunological processes.