Carbohydrate sequence analysis by electrospray ionization-mass spectrometry.
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Carbohydrate Sequence↗
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The amino-acid and carbohydrate sequences of a short glycopeptide isolated from bovine kappa-casein.
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Alkaline Phosphatase↗
The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development.
The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N-glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N-glycans containing a 3-fucosyl N-acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N-acetyllactosamine sequence [Lewisx (Le(x)) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.
Animals↗
Structure of the major neutral oligosaccharide-alditols released from the egg jelly coats of Axolotl maculatum. Characterization of the carbohydrate sequence GalNAc(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3/6).
Several O-linked oligosaccharides of the jelly coat surrounding the eggs of Axolotl maculatum were analysed by 1H-NMR spectroscopy. The four major oligosaccharidealditols released by reductive beta-elimination display either the Lewisx (Lex) determinant or the sequence GalNAc(beta 1-4)[Fuc(alpha 1-3)]GlcNAc. This last structure has previously been characterized in allergenically active oligosaccharides isolated from the sea squirt H-antigen, and in the N-linked glycans of Schistosoma mansoni and human urokinase. It represents the major carbohydrate chain found in A. maculatum, the oviduct of which constitutes an excellent source of beta 1-4-acetylgalactosaminyl transferase activity. Moreover, the carbohydrate chains isolated from A. maculatum are quite different from those found in seven other amphibian species, in which the presence of species-specific material has been characterized. The role of carbohydrates appears more and more apparent during the fertilization process, and the diversity of the O-linked oligosaccharides supports such a biological role.
Ambystoma↗
Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding.
Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.
Carbohydrate Sequence↗
Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole Ion trap mass spectrometer: neutral oligosaccharides and N-linked glycans.
Several oligosaccharide and N-linked glycan samples have been utilized to evaluate structural detail obtained with an ion trap mass spectrometer (ITMS). Using multistage MS/MS (MSn) in a commercial instrument, linear sequence, linkage, and branching have been studied, as well as the positional isomers within branched glycans. Samples were prepared as methylated derivatives, which amplifies the structurally informative cross-ring and double glycosidic cleavage products associated with interresidue linkage and oligomer branching detail. Spectral analysis of a linear glucose homopolymer demonstrated ITMS to be comparable with data obtained from a triple-quadrupole instrument, and in a study of complex glycans, the feature of MSn enabled the assignment of linkages that cannot be assessed by conventional triple-quadrupole tandem instrumentation. The longer time scale of CID experiments enhances the abundance of multiple bond ruptures important for a complete understanding of structure. This technology appears to provide a significant step toward the goal of characterizing all aspects of carbohydrate structure using a single instrument.
Carbohydrate Conformation↗
Congruent strategies for carbohydrate sequencing. 2. FragLib: an MSn spectral library.
A bottom-up approach to achieve full oligosaccharide and glycan characterization has been described that is based on an MSn fragment spectral library and associated tools. The library, identified as FragLib, was initiated with known standards and commercially available oligomers prepared as methylated derivatives. As a component of this effort a set of software tools has been written for storing, organizing, and comparing spectral files, including the identification of isobaric mixtures. These tools provide a facile and objective evaluation of structural details including interresidue linkage, monomer identification, anomeric configuration, and branching. The tools are components of a web-based data sharing interface for sample tracking, spectral searching, and structural confirmation. Applications have been detailed with unknown samples and previously characterized glycoconjugates.
Carbohydrate Sequence↗
The 9-O-acetylated disialosyl carbohydrate sequence of CDw60 is a marker on activated human B lymphocytes.
Gangliosides with a terminal 9-O-acetylated disialosyl group (CDw60 structures) show a restricted surface expression on human leukocytes. Hithereto, they have only been detected on subpopulations of human T lymphocytes. Using the defined CDw60 antibody UM4D4 and two new antibodies with preferential CDw60 activities, F6 and Z17, we demonstrate for the first time that CDw60 is an activation marker on human B lymphocytes. In vitro phorbol ester-stimulated human peripheral blood B lymphocytes as well as in vivo activated tonsillar B lymphocytes became CDw60 positive. CDw60 expression of these cells exceeds that of resting and activated T-lymphocytes.
Acetylation↗
Variation in the core and branch carbohydrate sequences of serum glycoprotein hormone alpha subunit as determined by lectin affinity chromatography.
Serum alpha subunits from patients with pituitary tumours and from normal controls were studied for their ability to bind to Lens culinaris agglutinin-Sepharose (LCA), L-phytohaemagglutinin-agarose (L-PHA) and soybean agglutinin-Sepharose (SBA). Serum alpha subunits from normal controls which had previously been shown to bind to Concanavalin A-Sepharose (Con A) were not retained by LCA. In contrast, Con A-reactive alpha subunits from patients with pituitary tumours bound specifically to LCA. Non-Con A-reactive alpha subunits from patients with pituitary tumours were also largely not bound to LCA, but were retained by L-PHA. No alpha subunits from any source bound to SBA. These results indicate that the structural alterations resulting in non-Con A-reactive serum alpha subunits include highly branched complex oligosaccharides in addition to the hybrid-type glycans previously described. The increased branching appears to be associated with fucosylation in the core region of the oligosaccharides. Serum alpha subunit from any source appears to be devoid of terminal N-acetylgalactosamine residues. These structural modifications may be related to the variable biological activity of alpha subunit which has been reported.
Carbohydrate Sequence↗
Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes.
We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.
Adult↗
Structure of three Kdn-containing oligosaccharide-alditols released from oviducal secretions of Ambystoma tigrinum: characterization of the carbohydrate sequence Fuc (alpha 1-5) [Fuc (alpha 1-4)] Kdn (alpha 2-3/6).
Oligosaccharide-alditols released by reductive beta-elimination from the egg jelly coat of Ambystoma tigrinum were analyzed by 1H NMR spectroscopy. As observed for five other amphibian species, these carbohydrate chains are highly species-specific, and should support the species-specificity of gamete interaction. The carbohydrate chains of Ambystoma tigrinum are characterized by the presence of a new type of sequence: Fuc (alpha 1-5) [(Fuc (alpha 1-4)] Kdn (alpha 2-3/6).
Ambystoma↗
Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc beta 1-4Gal found in some glycolipids.
Pneumonia is one of the most common causes of death from infectious disease in the United States. To examine the possible role of carbohydrates as adhesion receptors for infection, several pulmonary pathogenic bacteria were studied for binding to glycosphingolipids. Radiolabeled bacteria were layered on thin-layer chromatograms of separated glycosphingolipids, and bound bacteria were detected by autoradiography. The classic triad of infectious bacteria found in cystic fibrosis, Pseudomonas aeruginosa, Haemophilus influenzae, and Staphylococcus aureus, along with other bacteria commonly implicated in typical pneumonia, such as Streptococcus pneumoniae, Klebsiella pneumoniae, and certain Escherichia coli, bind specifically to fucosylasialo-GM1 (Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Cer), asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta-1-4Galc beta 1-1Cer), and asialo-GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer). Bacteria maintained in nutrient medium bind better than the same cells suspended in buffer. They do not bind to galactosylceramide, glucosylceramide, lactosylceramide, trihexosylceramide, globoside, paragloboside, Forssman glycosphingolipid, or several other glycosphingolipids tested, including the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and Cad. The finding that these pathogens do not bind to lactosylceramide suggests that beta 1-4-linked GalNAc, which is positioned internally in fucosylasialo-GM1 and asialo-GM1 and terminally in asialo-GM2, is required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient for binding, as the bacteria did not bind to globoside, which contains the terminal sequence GalNAc beta 1-3Gal. These data suggest that these bacteria require at least terminal or internal GalNAc beta 1-4Gal sequences unsubstituted with sialyl residues for binding. Other bacteria, including Mycoplasma pneumoniae, Streptococcus pyogenes, Salmonella species, and some E. coli, do not bind to the GalNAc beta 1-4Gal sequence. The biological relevance of these data is suggested by our finding that substantial amounts of asialo-GM1 occur in human lung tissue.
Animals↗
Structural similarity of the terminal carbohydrate sequences of glycoproteins and glycolipids.
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Acetylglucosamine↗
The carbohydrate sequence of the glycopeptide chains of human transferrin.
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Asparagine↗
Primary sequence analysis and representation techniques in carbohydrates.
Sequence similarity calculations of carbohydrates present several problems which must be addressed if a computer implementation is to be achieved. These problems range from the computational representation of the complex carbohydrate structure to the method by which the comparison of residue and linkage is to be made. This paper therefore discusses the form of this representation and how two or more carbohydrates can be meaningful compared. An example set of results using this approach is presented and discussed to illustrate how similarity comparison can show relationships between carbohydrates, features that are otherwise hidden by the sheer volume of data which must be considered.
Algorithms↗
Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates.
Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.
Carbohydrate Sequence↗
Distribution of (fucogalactosyl-)epitope expressing glycoconjugates in rat brain.
Glycoconjugates are known to be concentrated in plasma membranes, especially in synaptic junctions, where they subserve various functions in neural connectivity. Here we report the cellular distribution of a new monoclonal antibody recognizing (fucogalactosyl) sequences in carbohydrate structures. The most pronounced immunoreactivity was found in fibrous astrocytes, in many parts of the brain and with lower density in various neuronal elements. This points to the expression of identical carbohydrate sequences on molecules within certain glial and neuronal elements. Previous intracerebral injections of the antibody interfered with long term memory formation. Therefore, functions mediated by corresponding glycoproteins in neurons and glia cells or even neuron-glial interactions, might be relevant for information-processing.
Animals↗
Structure determination of the octa- and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestinal heparin.
Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G. (1992) J. Biol. Chem. 267, 1528-1533). In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only. IV was successfully isolated only after subsequent digestion with glycuronate-2-sulfatase. Their structures were determined by chemical and enzymatic analyses and 1H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: delta HexA(2S)alpha 1-4GlcN(NS,6S)alpha 1-4GlcA beta 1-4GlcNAc alpha 1-4- GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (I), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (II), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1- 4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcNAc-alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (III), delta HexA alpha 1-4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc(6S)alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (IV) (delta HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II contained 1 mol of Gly in addition to Ser. The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures. The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin.
Amino Acids↗