Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

The presence of an endogenous lectin in early embryos ofXenopus laevis

Summary Xenopus laevis embryos were examined for the presence of endogenous carbohydrate binding proteins. Soluble extracts of cleavage, gastrula and neurula embryos are able to agglutinate trypsinized rabbit erythrocytes. Unlike other embryonic lectins this agglutination activity requires the presence of calcium ions but not of sulphydryl reducing agents. It is specifically inhibited by galactose and galactose containing derivatives. Thiodigalactoside is the most potent disaccharide inhibitor followed by lactose and melibiose respectively. Methyl -d-galactopyranoside is a more effective inhibitor than its anomer. N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, methyl -d-mannopyranoside andl-fucose do not inhibit activity at concentrations at or above 25 mM. EDTA and EGTA are also strong inhibitors of this activity. -d-galactoside binding lectins present in the early chick embryo have been implicated in cell to cell and cell to substrate adhesiveness of extraembryonic chick endoderm cells. Since cells of the blastula inXenopus laevis possess surface receptors bearing terminal -d-galactoside groups it is possible that this -d-galactoside binding lectin may play a role in the control of cell surface mediated events during development.     

A method for glycoconjugate synthesis

7-Formylheptyl glycosides of 2-acetamido-2-deoxy--d-glucopyranose andO--l-rhamnopyranosyl-(1 3)-O--l-rhamnopyranose were synthesized and were coupled by reductive amination to bovine serum albumin and aminopropyl glass, respectively.     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

Synthesis of deoxygalactose-containing sialyl LeX ganglioside analogues to elucidate the structure necessary for selectin recognition

Sialyl Lewis X ganglioside analogues containing 4-deoxy-, 6-deoxy-, and 4,6-dideoxy-d-galactopyranose in place ofd-galactopyranose have been synthesized. Glycosylations of 2-(trimethylsilyl)ethyl 2,6-di-O-benzyl--d-galactopyranoside and 2-(trimethylsilyl)ethyl -d-fucopyranoside with the phenyl 2-thioglycoside derivative of sialic acid, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) as the promoter in acetonitrile, gave the desired 2-(trimethylsilyl)ethyl sialyl--(23)--d-galactopyranoside and--d-fucopyranoside, respectively. The sialylgalactose derivative obtained was then modified to 4-deoxy and 4,6-dideoxy derivatives. These were converted, byO-benzoylation, transformation of the 2-(trimethylsilyl)ethyl group to trichloroacetimidates, and introduction of the methylthio group with methylthiomethysilane, into the corresponding glycosyl donors, which were then coupled with 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(13)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(13)-2,4,6- tri-O-benzyl--d-galactopyranoside in the presence of dimethyl(methylthio)sulfonium triflate (DMTST). The resulting pentasaccharides were each converted to the corresponding -trichloroacetimidates, which, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the desired sphingosine derivatives. Selective reduction of the azide group,N-acylation with octadecanoic acid,O-deacylation, and saponification of the methyl ester afforded the target compounds.Synthetic Studies on Sialoglycoconjugates, Part 79.     

Fucosyltransferase expression in human platelets and leucocytes

The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Synthesis of an H type 2 and a Y (Ley) glycoside from thioglycoside intermediates

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 1 and the tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-3-O-(-l-fucopyranosyl)-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 2 were synthesized. Thioglycosides, suitably protected, activated directly with methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfonium tetrafluoroborate or activated after bromine treatment with halophilic reagents, were used as glycosyl donors in the construction of the glycosidic linkages.Abbreviations DMTSB dimethyl(methylthio)sulfonium tetrafluoroborate - Phth phthaloyl - MBn p-methoxybenzyl - ClBn p-chlorobenzyl     

Mikrochemische Untersuchung der α-d-Glucosidasen mit 4-Methylumbelliferyl-und 2-Naphthyl-α-d-glucosid

Zusammenfassung In Rohhomogenaten aus gefriergetrockneten Kryostat-schnitten von verschiedenen Rattenorganen werden die K _m und V _max der neutralen und sauren -d-Glucosidase bestimmt und der Einfluß von pH, Substrat- und Enzymkonzentration und Inkubationszeit auf die Aktivität fluorometrisch mit 4-Methylumbelliferyl-und 2-Naphthyl--d-glucosid als Substraten ermittelt.Mit den biochemischen Daten werden 2 mikrochemische Ansätze zur fluorometrischen Messung dieser Glykosidasen entwickelt und die saure und neutrale -Glucosidase in Gruppen von Epithelzellen nach Isolierung aus gefriergetrockneten Kryostatschnitten von Nebenhoden, Jejunum, Ilium, Niere und Leber untersucht. Im Vergleich zum 2-Naphthylderivat sind beide -Glucosidasen mit 4-Methylumbelliferyl--d-glucosid weniger aktiv. Allerdings fluoresziert 4-Methylumbelliferon etwa 100mal intensiver als 2-Naphthol, so daß das Methylumbelliferonderivat zur Messung der -Glucosidasen speziell in schwach aktiven Zellen der 2-Naphthylverbindung vorzuziehen ist.

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Microchemical investigation of -d-glucosidases using 4-methylumbelliferyl-and 2-naphthyl--d-glucoside

Summary In crude homogenates prepared from freeze-dried cryostate sections of various rat organs the K _m and V _max of acid and neutral -glucosidase as well as the effect of the pH, substrate and enzyme concentration and the incubation time on the activity were determined fluorometrically with 4-methylumbelliferyl-and 2-naphthyl -d-glucoside as substrates.On the basis of the biochemical data 2 assays were developed for the microchemical measurement of both -glucosidases in groups of epithelial cells isolated from freeze-dried cryostate sections of the epididymis, jejunum, ilium, liver and kidney of suckling and adult rats. The rate of hydrolysis of 2-naphthyl and 4-methylumbelliferyl -d-glucoside differs moderately. However, due to the higher sensitivity of 4-methylumbelliferone the methylumbelliferyl derivative is preferable especially for the evaluation of -d-glucosidases in cells with low enzyme activity.

     

Characterization of the specificity of binding ofMoluccella laevis lectin to glycosphingolipids

The specificity ofMoluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal -linkedN-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc3GalNAc3Gal4Gal4Glc1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc3(Fuc2)Gal. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal -linked galactose. When considering the use of theM. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance. Nomenclature: The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for Lipids:Eur J Biochem (1977)79:11–21,J Biol Chem (1982)257:3347–51, andJ Biol Chem (1987)262:13–18). It is assumed that Gal, Glc, GlcNAc, GalNAc, and NeuAc are of thed-configuration, Fuc of thel-configuration, and all sugars present in the pyranose form.     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry

A novel lectin from the mushroom Marasmius oreades (MOA) has been shown to bind to human blood group B oligosaccharides [1]. In the present work we examine the binding of a series of analogues of the blood group B-trisaccharide, Gal(1-3)[Fuc(1-2)]Gal-OR (1, R = (CH₂)₈COOMe). MOA was biotinylated and immobilized on a micro column (9.8 L) for evaluation by Frontal Affinity Chromatography-Mass Spectrometry (FAC-MS) [2]. The trisaccharide 1 was found to be the epitope needed for maximum recognition (K _d = 3.6 M). A series of synthetic deoxygenated and O-methylated analogues of the B-trisaccharide (R = OMe) were then screened against the lectin, and the key structural elements for binding were determined. OH-4 of the -Gal residue and OH-2 of the -Gal residue were found to be critical for recognition. The FAC-MS technique also proved powerful in evaluating mixtures of compounds. Since the solution NMR structure and crystal structure of the B-trisaccharide are known [3], we propose the specific surface of the trisaccharide that is recognized by the lectin. Published in 2003.     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

Anomeric preferences ofd-glucose uptake and utilization by cerebral cortex slices of rats

On aerobic incubation of rat cerebral cortex slices with anomers ofd-glucose and with 2-deoxy-d-glucose (2DG) for 5 min, the disappearance of -d-glucose from the incubation mixture was greater than that of -d-glucose and both anomers had a greater rate of disappearance than that of 2DG. In addition, there were significantly greater consumption of oxygen and production of lactate with the -anomer than with the -anomer. In similar experiments with³H-labeledd-glucose anomers and [1-³H]-3-O-methyl-d-glucose (3MG), the accumulation of [1-³H]--d-glucose (up to 5 min) by rat cerebral cortex slices was greater than that of [1-³H]--d-glucose. Although initially lower than that of the anomers, the accumulation of [1-³H]-3MG increased at a greater rate and, by 5 min of incubation, was greater than that of both glucose anomers. This preferential accumulation was seen to disappear when the slices were preincubated with 2DG (hexokinase inhibitor) or when the temperature of incubation was reduced to 20°C. Under those conditions the data with the glucose anomers were similar to those obtained with 3MG. Our data then suggested that the greater accumulation of -d-glucose than of -d-glucose by the slices was probably not due to differences in transport through brain cell membranes but rather to the preferential metabolism of the -d-glucose.     

Structure and properties of aCephalosporium acremonium α-galactosidase

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified -galactosidase (EC 3.2.1.22) from the moldCephalosporium acremonium has been studied. The results obtained by gas liquid chromatography indicated the presence ofN-acetylglucosamine, mannose, galactose andN-acetylneuramic acid in the molar proportions 27311. The presence of two types of Asn-linked oligosaccharide structures in the enzyme molecule is assumed. The -galactosidase liberates (1–3), (1–4) and (1–6)-linkedd-galactose units from various synthetic and natural substrates which have been tested. The effects of pH, substrate concentration and temperature on the catalytic activity of the enzyme are described. The purified -galactosidase also exhibited a lectin activity with an affinity towards glucose, and to some extent mannose.Abbreviations p-NPG p-nitrophenyl--d-galactopyranoside - 4-MUG 4-methylumbelliferyl--d-galactopyranoside - HU hemagglutinin unit - PBS phosphate buffered saline - SDS sodium dodecyl sulphate - ConA Concanavalin A - WGA wheat germ agglutinin - LCA Lens culinaris agglutinin - PHA phytohemagglutinin fromPhaseolus vulgaris     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [¹⁴C]glucans using either guanosine 5-diphosphate (GDP)-D-[U-¹⁴C]glucose or uridine 5-diphosphate (UDP)-D-[U-¹⁴C]glucose as glycosyl donors. Although these glucans had -(13) and -(14) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed -(13) and -(14) glucan from GDP-D-[U-¹⁴C]glucose was changed to that of -(14) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-¹⁴C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K _m and V _max of the glucan synthase for GDP-D-glucose were 6.38 M and 5.08 M·min^-1, respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.Abbreviations GDP guanosine 5-diphosphate - GLC gasliquid chromatography - UDP trridine 5-diphosphate