Imaging single glycans.

Imaging of biomolecules guides our understanding of their diverse structures and functions1,2. Real-space imaging at sub-nanometre resolution using cryo-electron microscopy has provided key insights into proteins and their assemblies3,4. Direct molecular imaging of glycans-the predominant biopolymers on Earth, with a plethora of structural and biological functions5-has not been possible so far6. The inherent glycan complexity and backbone flexibility require single-molecule approaches for real-space imaging. At present, glycan characterization often relies on a combination of mass spectrometry and nuclear magnetic resonance imaging to provide insights into size, sequence, branching and connectivity, and therefore requires structure reconstruction from indirect information7-9. Here we show direct imaging of single glycan molecules that are isolated by mass-selective, soft-landing electrospray ion beam deposition and imaged by low-temperature scanning tunnelling microscopy10. The sub-nanometre resolution of the technique enables the visualization of glycan connectivity and discrimination between regioisomers. Direct glycan imaging is an important step towards a better understanding of the structure of carbohydrates.

Phenolic Compounds, Volatiles and Antioxidant Capacity of White Myrtle Berry Liqueurs.

The aim of this research was to evaluate the antioxidant capacity and physical-chemical characteristics of commercial white myrtle berry (Myrtus communis L. var. leucocarpa DC) liqueur (WMBL). The total phenolic (TP) content was measured spectrophotometrically, applying a modified Folin-Ciocalteu's method, and phenolic compounds were identified by high-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry, and quantified by HPLC coupled with ultraviolet/visible detection. The antioxidant capacities were evaluated by FRAP, CUPRAC, DPPH•, and ABTS•+ assays. The volatiles were assessed by gas chromatography and mass spectrometry (GC-MS/FID) after headspace solid-phase microextraction (HS-SPME) and liquid-liquid extraction (LLE). WMBL showed lower TP levels (636.3 ± 39.2 mg GAE/L) than in purple myrtle berry liqueur (PMBL). Nevertheless, WMBL exhibited better antioxidant capacities, potentially due to high concentrations of gallic acid (294.2 ± 14.2 mg/L) and its derivatives (58.3 ± 2.1 mg/L). Other phenolic compounds detected by HPLC-DAD and LC-MS/MS were flavonols like myricetin and its derivatives (myricetin-3-O-galactoside and myricetin-3-O-rhamnoside) with concentrations similar to those found in PMBL. GC-MS/FID analysis revealed 44 compounds (terpenes, higher aliphatic compounds and shikimic acid pathway derivatives). 1,8-Cineole was the most abundant terpene in the liqueur (26.5% (HS-SPME) and 9.6% (LLE)).

Changes in the major cell envelope components of Mycobacterium tuberculosis during in vitro growth.

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

Determination of Afzelin and Astragalin from Lespedeza cuneata on Aldose Reductase Inhibition.

The bioactive chemicals in L. cuneata were investigated by repeated column chromatography and their effect on aldose reductase (AR), obtained from rat lenses, was examined. Results showed that the ethyl acetate and n-butanol fractions of L. cuneata exhibited potential inhibitory effect against AR with IC50 values of 0.57 and 0.49 µg/mL, respectively. Phytochemical analysis of these two fractions resulted in the isolation of five flavonoids namely, acacetin (1), afzelin (2), astragalin (3), kaempferol (4) and scutellarein 7-O-glucoside (5). The AR inhibitory effect of compounds 1-5 was explored; compounds 2, 3 and 5 showed potential AR-inhibitory effects with IC50 values of 2.20, 1.91 and 12.87 µM, respectively. Quantitative analysis of afzelin (2) and astragalin (3) in L. cuneata by high performance liquid chromatography with ultraviolet detection revealed its content to be 0.722-11.828 and 2.054-7.006 mg/g, respectively. Overall, this study showed that L. cuneata is rich in flavonoids with promising AR-inhibitory activities, which can be utilized for the development of natural therapies for treating and managing diabetic complications.

Inter-population and inter-organ distribution of the main polyphenolic compounds of Epilobium angustifolium.

Rosebay willowherb (Epilobium angustifolium) contains large amounts of polyphenolic compounds, including tellimagrandin I-based oligomeric ellagitannins (ETs). The aim of this study was to assess the interpopulational and inter-organ variability of the polyphenol fingerprint of E. angustifolium. Seven ETs, 11 flavonol glycosides and neochlorogenic acid were quantified by UHPLC-DAD-ESI-QqQ-MS in the leaves, flowers and stem parts of plants from 10 populations. Total polyphenol content of leaves and flowers ranged from 150 to 200 mg/g dry wt, of which 90% was constituted by dimeric to heptameric ETs. Flowers contained, on average, 10% more oenothein B (dimeric ET) and 2 times less oenothein A (trimeric ET) than leaves. Tetrameric and pentameric ETs exhibited rather similar levels in leaves and flowers whereas hexameric and heptameric were 3-4 times more abundant in flowers than in leaves. Quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside were specific to flower tissue and were absent from leaves. The inflorescence stem showed the highest content in total polyphenols with an average of 250 mg/g dry wt and contained remarkably large amounts of oenothein B and A. Polyphenol content steadily decreased along the inflorescence stem and reached its lowest level in the vegetative part of the stem. The interpopulational variability of most polyphenols was within a two- to threefold range across the 10 sampled populations. Myricetin-3-O-glucoside and myricetin-3-O-glucuronide, however, showed a more population-specific distribution with concentrations varying from 0 to 2.3 mg/g dry wt. Finally, this study showed that the levels of oenothein B and A in the plant are not interdependent but that their relative abundance is constant within a population.

Efficient installation of beta-mannosides using a dehydrative coupling strategy.

[reaction: see text]. A new coupling procedure for the construction of the challenging beta-mannosidic bond is described. Dehydrative mannosylation using 4,6-O-benzylidene mannopyranoses allows for the formation of beta-mannosides in excellent yield. The stereoselectivity is generally good but influenced by the exact nature of the glycosylating agent and the nucleophile.

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches.

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Man5GlcNAc and related free oligomannosides in the cytosol fraction of hen oviduct.

The presence of free oligomannosides in cytosol has been demonstrated by metabolic radiolabeling, and Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc was detected as the main free oligomannoside in Chinese hamster ovary cells [Kmiécik, D., Herman, V., Stroop, C.J.M., Michalski, J.C., Mir, A.M., Labiau, O., Verbert, A., and Cacan, R. (1995) Glycobiology, 5, 483-494]. In the present paper, the isomeric structures and amounts of oligomannosides in the cytosol fraction of hen oviduct were analyzed by pyridylamination and exoglycosidase digestion. Hen oviduct was used since our group has already characterized the cytosolic neutral alpha-mannosidase and endo-beta-N-acetylglucosaminidase obtained from the same source. The amounts of Man2GlcNAc, Man3GlcNAc, Man4GlcNAc, and Man5GlcNAc were 0.6, 0.6, 0.5, and 0.8 nmol/g tissue, respectively, but Man6GlcNAc-Man9GlcNAc were not detected. The isomeric structures of the Man3GlcNAc-Man5GlcNAc found were (Manalpha1-2)0-2Manalpha1-3(Manalpha1-6)Manbeta1 -4GlcNAc, which were compatible with the substrate specificities of cytosolic endo-beta-N-acetylglucosaminidase and neutral alpha-mannosidase, indicating that these enzymes participate in the formation of the oligomannosides present in the cytosol.

A histochemical study of inflammatory lesions of the maxillary sinus mucosa using biotinylated lectins.

The distribution of glycoconjugates in normal maxillary sinus tissues, in cases of maxillary sinusitis and in postoperative maxillary cysts (POMC), was examined using seven different lectins as probes. The results showed that wheatgerm agglutinin (WGA), peanut agglutinin (PNA), Ulex europaeus agglutinin-1 (UEA-1), Ricinus communis agglutinin-1 (RCA-1), and concanavalin A (ConA) strongly react with the cilia and goblet cells. The binding of WGA, PNA, UEA-1, and RCA-1 was increased in maxillary sinusitis and POMC compared with normal maxillary sinus epithelium, whereas that of ConA was decreased. The decreased binding of ConA suggested that there were fewer mannoside residues in the maxillary sinus epithelium in the inflammatory lesion. The PNA bound to the cilia, goblet cells and mucous glandular cells in maxillary sinusitis and POMC, but not in normal, uninflamed cells, indicating that D-galactose was produced by the inflammatory condition. Similar binding patterns of PNA and RCA-1 were found in the cilia and on the surface of the epithelium and in the goblet cells. It is assumed that the carbohydrate moiety in the sinus mucosa is altered in inflammatory conditions.

Reversed-phase high-performance liquid chromatography of the cardiac glycoside LNF-209 with refractive index detection.

LNF-209 is a glycoside, similar to digoxin, which has potential for use in the treatment of congestive heart failure. However, unlike digoxin it exhibits virtually no useful UV absorption spectra, making detection difficult. One means of detection is the refractive index detector, but like most bulk property detectors it has certain limitations. Its sensitivity is limited and it is sensitive to small changes in a number of parameters, such as temperature, mobile phase composition, and flow-rate. These parameters must be closely controlled to obtain a stable baseline. This paper describes the steps taken to control the system and the development and validation of an assay for LNF-209 in dosing solutions. The method developed is capable of quantitating LNF-209 in solutions of sterile water and 5% dextrose at concentrations ranging from 8 to 6000 micrograms/ml. The method is linear over this range and quantitative recovery is obtained. The overall average relative standard deviation for replicate analysis of several samples at various concentrations assayed over two days was 2.3%.

Onset and regression of neuroaxonal lesions in sheep with mannosidosis induced experimentally with swainsonine.

A group of young sheep were fed a diet containing the alpha-mannosidase inhibitor swainsonine, which resulted in the induction of a neuronal lysosomal mannoside storage disease. Sheep were killed at various intervals during and following the treatment period and the nature and distribution of neuronal and axonal lesions in the brain were assessed by routine light and electron microscopy and by the rapid Golgi impregnation technique. Neuronal mannoside storage, axonal dystrophy and meganeurite formation were induced by 80 days of treatment and the lesions had regressed by 40 days after the end of treatment. The results are discussed in relation to their relevance to the current widespread interest in the pathobiology of neuronal lysosomal storage.

Peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).

Kinetics of alpha-mannosidase action on various alpha-D-mannopyranosyl linkages in hen ovalbumin glycopeptides as monitored by carbon 13 nuclear magnetic resonance spectroscopy.

We show that 13C NMR spectroscopy is a practical method for following the kinetics of enzymatic digestion of individual carbohydrate residues of glycopeptides and for determining the structures of the products of partial digestions. Specifically, we study the jack bean alpha-mannosidase digestion of the two hen ovalbumin glycopeptides Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound A) and Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound B). The reported "rule" that jack bean alpha-mannosidase hydrolyzes Man alpha 1 leads to 2Man and Man alpha 1 leads to 6Man linkages at least 15 times faster than Man alpha 1 leads to 3Man linkages (Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J. Biol. Chem. 250, 8569-8575) is not of general validity. Although the Man alpha 1 leads to 2Man (alpha) linkage of Compound A is the first one to be digested, the Man alpha 1 leads to 3Man (beta) linkage is hydrolyzed next, faster than the Man alpha 1 leads to 6Man (alpha) linkage. The Man alpha 1 leads to 3Man (alpha) linkage is hydrolyzed very slowly. We show that it is practical to use 13C NMR spectroscopy to determine when the enzymatic digestion should be halted to isolate derivatives such as Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn and Man alpha 1 leads to 3Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn.

Frontal affinity chromatography of ovalbumin glycoasparagines on a concanavalin A-sepharose column. A quantitative study of the binding specificity of the lectin.

The interactions of Sepharose 4B-immobilized concanavalin A (ConA) with 10 glycoasparagines derived from ovalbumin were investigated quantitatively by frontal affinity chromatography. In this method, a carbohydrate solution is applied continuously to a ConA-Sepharose column and the retardation of the elution front is measured as a parameter of the strength of the interaction. The dissociation constant (Kd) for each saccharide with ConA can be determined. An analysis of the binding of p-nitrophenyl-alpha,D-mannoside has shown that the binding properties of ConA do not change essentially after immobilization on Sepharose 4B. Each of the ovalbumin glycoasparagines was labeled with tritium by the reductive methylation method for analysis. A comparison of the Kd values obtained showed that the binding of ConA varies considerably with very slight structural differences of the glycosyl chain. The results suggest that ConA recognizes a specific glycosyl chain structure, Man alpha 1-6(Man alpha 1-3)Man, in which at least one hydroxyl group at the C-3 position of C-6-linked mannose should be free. The glycoasparagines containing this structure bound strongly to ConA-Sepharose with dissociation constants below 3.4 X 10(-7) M.

Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit.

Chemical mapping of the luminal surface of normal rabbit aortic and coronary endothelium was investigated cytochemically to establish a baseline for further comparison with the biochemical changes possibly induced by the experimental hypercholesterolemia. Morphometric analysis showed that in the aortic endothelium the plasma membrane exposes a large number of uniformly-distributed positively-charged groups of high pKa, and a heterogeneous pattern of dense anionic groups of low pKa. Among the latter, only a third was represented by neuraminidase-cleavable sialic acids. These are constituted by various classes of N-, and O-substituted sialyl residues in glycoconjugates, most frequent being those non-O-acetylated at C8 or C9. Among the oligosaccharides detected with lectins, very abundant were the glycoconjugates containing mannosyl and subterminal galactosyl, whereas N-acetyl-glucosamine, terminal galactosyl and N-acetyl-galactosaminyl moieties were rather poorly represented. The density of the latter two markedly increased after its unmasking by neuraminidase treatment. Coated pits contained both anionic and cationic sites, but only few sialic acids and saccharide residues in significantly lower amounts than plasma membrane. The membrane of plasmalemmal vesicles displayed a high number of cationic sites and mannosyl residues, but very few anionic groups, sialyl residues, and galactosyl and N-acetyl-galactosaminyl moieties. Coronary endothelium displayed a chemical pattern similar to aorta, with some differences, especially in the frequency of some oligosaccharides. Vena cava was low in acidic groups but rather rich in galactose. Plasmalemmal vesicles were only occasionally labeled by the probes used. Monocyte surface exhibited a high density of anionic sites, and binding sites for wheat germ agglutinin and Ricinus communis agglutinin. No mononuclear cells were observed adhering to endothelial surface.

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha-D-mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X-ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide-free concanavalin A. The structure of the saccharide complex was refined by restrained least-squares methods to an R-factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha-D-mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site. A network of seven hydrogen bonds connects oxygen atoms O-3, O-4, O-5 and O-6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O-2 and O-1 of the mannoside extend into the solvent. O-2 is hydrogen-bonded through a water molecule to an adjacent asymmetric unit. O-1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

Glycosylphosphatidylinositols of Plasmodium chabaudi chabaudi: a basis for the study of malarial glycolipid toxins in a rodent model.

Free and protein-bound glycosylphosphatidylinositols (GPIs) of the blood stages of the rodent malarial parasite Plasmodium chabaudi chabaudi AS were identified and characterized. TLC analysis of material extracted by organic solvents from metabolically labelled parasites revealed a distinct set of glycolipids. These glycolipids were identified as GPIs by specific chemical and enzymic treatments and by structural analysis of their glycan and hydrophobic parts. These analyses revealed that P.c.chabaudi AS synthesizes a set of GPI-biosynthesis intermediates and two potential GPI-anchor precursors exhibiting the following structures: ethanolamine-phosphate [(alpha1-2)mannose]mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine - (acyl) inositol-phosphate-diacylglycerol (P.ch. alpha) and ethanolamine-phosphate - mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine-(acyl)inositol-phosphate-diacylglycerol (P.ch. beta). One of these GPI-anchor precursors (P.ch. alpha) possesses the same carbohydrate structure as the GPI membrane anchor of merozoite surface protein-1 from P.c.chabaudi AS.