Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Molecular cloning and sequencing analysis of a β-tubulin gene from Lupinus albus

Genomic -Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the -tubulin cDNA ( 8–31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the -tubulin gene, designated as L1 and identified by similarity with other -tubulins. The L1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg²⁺. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus -tubulin genes are constitutively expressed in all studied plant tissues.     

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

UDP-GlcNAc:GlcNAc 1-2Man1-6R (GlcNAc to Man) 1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc1-2Man1-6Glc/Man-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man1-3 residue and the 4-hydroxyl of the Man- residue of the Man1-6(Man1-3)Man-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAc1-2(4,6-di-O-methyl-)Man1-6Glc-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.Abbreviations all allyl - AML acute myeloid leukaemia - BSA bovine serum albumin - CML chronic myelogenous leukaemia - Gal G,d-galactose - Glc d-glucose - GlcNAc Gn,N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man M,d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈COOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - oct octyl - pnp p-nitrophenyl - T transferase     

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Gal1-4GlcNAc1-2[Gal1-4GlcNAc1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Gal1-4GlcNAc1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either (2–6)-linkedN-acetylneuraminic acid or (1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Gal1-4GlcNAc1-6Man1-6 branch, respectively. The same substitutions on the Gal1-4GlcNAc1-6Man1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAc2-6 and Fuc1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Man1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.Abbreviations L-PHA leukoagglutinating phytohemagglutinin - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - GP glycopeptide - OS oligosaccharide - HPLC high-performance liquid chromatography - FNR fraction not retarded - FR fraction retarded suffixes MS, BS and TS indicate mono-, bi- and trisialyl derivatives respectively; suffix MF indicates monofucosyl derivatives.structures of the substratesOS2, OS3, OS3, OS4, GP2, GP3, GP4, GP4-MF, OS2(3) andOS2(-) are presented in Fig. 2     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

The tryptic peptide composition of the beta chains of hemoglobins A and B of the domestic cat (Felis catus)

Summary The tryptic peptides from the ^A and ^B chains of cat hemoglobins A and B have been isolated and the amino acid compositions determined. Differences between the two chains were found in two peptides,T-1 (GlySer) andT-14 (AsnSer and LysArg). The GlySer and LysArg substitutions are placed at-1 and-144 respectively from earlier work, and the third substitution, AsnSer at-139 is suggested from this work. In addition, the presence of a blocked amino terminus in ^B has been confirmed. Tentative sequences constructed by homology with known-chain structures suggest the occurrence of substitutions at _{1 1} contacts in ^A and ^B that may be functionally significant. There are at least 18 differences in amino acid composition between cat ^A and dog-chains and 22 differences between cat ^A and normal adult human-chains.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Regioselective deacetylation of peracetylated monosaccharide derivatives by polyethylene glycol-modified lipase for the oligosaccharide synthesis

Lipase modified with polyethylene glycol became soluble and active in organic solvents, and catalyzed regioselective deacetylation of peracetylated monosaccharide derivatives in 1,1,1-trichloroethane. The deacetylation occurred only at the positions of C-4 and C-6 of the glycopyranoside ring. Especially, peracetylated methyl -D-xylopyranoside and peracetylated L-serine--D-xylopyranoside were hydrolyzed only at the position of C-4. Subsequently, one of the resulting products, that is L-serine-2,3-di-O-acetyl--D-xylopyranoside, was coupled with galactose residue to obtain L-serine-4-O-(-D-galactopyranosyl)--D-xylopyranoside, a model compound of the carbohydrate-protein linkage region of proteoglycans.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Sinapoylglucose: malate sinapoyltransferase activity in seeds and seedlings of rape

Protein preparations from seeds and seedlings (cotyledons) of rape (Brassica napus subsp. napus [L.] DC.) catalyzed the transfer of sinapic acid from 1-Osinapoyl--glucose to malate in the formation of O-s-inapoylmalate. The enzyme involved, 1-O-sinapoyl--glucose: l-malate O-sinapoyltransferase (SMT; EC 2.3.1), catalyzes the key step in the overall conversion of the seed constituent sinapine (O-sinapoylcholine) to the accumulating O-sinapoylmalate by way of the intermediate 1-O-sinapoyl--glucose. The present paper describes this phenomenon focussing on SMT activity.Abbreviations Sin-Glc 1-O-sinapoyl--glucose - Sin-Mal O-sinapoylmalate - SMT 1-O-sinapoyl--glucose: l-malate sinapoyltransferase (EC 2.3.1) This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the Ontario Ministry of Agriculture and Food.     

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     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Antiinsectan properties of a novel Fusarium-derived sterol sulfate

The antiinsectan properties of the Fusarium graminearum-derived novel sterol sulfate, 4,4,24-trimethylcholesta-8,14,24(28)-trien-2,3,11,12-tetrol 12 acetate, 3-sulfate were tested on the corn earworm (Heliothis zea (Boddie), the fall armyworm (Spodoptera frugiperda (J. E. Smith), and the driedfruit beetle (Carpophilus hemipterus L.). The sterol sulfate could not be used as a sterol source by any of the insects. The sterol sulfate inhibited growth of S. frugiperda and C. hemipterus larvae at 2500 and 4000 ppm, respectively. Defensive implications of this compound are discussed.