Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

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.     

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.     

Comparative structural analysis of snail galactans by a radioimmunoassay to elucidate species-specific determinants

Summary Galactans, the storage polysaccharides in the perivitelline fluid of many snails showed a high degree of species-specificity as revealed by quantitative precipitin formations with lectins, polyclonal antisera, myeloma proteins as well as by the reactivity with the enzyme galactose oxidase. However, their chemical compositions were remarkably similar since thed-Gal residues were all linked 13 and 16 glycosidically.The specificity seemed to be related to the different degrees of branching in the various galactans but could also be due to some other minor constituents in some galactans such asl-galactose or phosphate.In this study a Radioimmunoassay was developed using the galactan of the snailLymnaea stagnalis to elucidate those differences which were only related to a unique distribution of the 13 and 16 linkages, since this galactan was composed exclusively ofd-galactose residues. The galactan was labeled by sequential oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Inhibition of the binding of the labeled galactan to insolubilized antibodies was investigated by galactans of different species, their chemically modified products, andd-galactose-composed oligosaccharides of unambiguously identified structures.Inhibition byLymnaea stagnalis galactan was about 45 000 times that ofHelix pomatia galactan. The most complementary oligosaccharide found was -d-Gal13[-d-Gal16]-d-Gal11l-Gro, being about 200 times more effective thand-Gal. However, a fraction with molecular weights between 700 and 1000 isolated from the partially hydrolized galactan was still seven times more effective. From the reactivity of the antiserum with the different oligosaccharides tested the following structure was inferred which most likely represented the complete determinant recognized by the antiserum: -d-Gal13[-d-Gal16]-d-Gal16[-d-Gal13]-d-Gal1. This determinant seemed to be most common inLymnaea stagnalis galactan and its frequency of occurrence appears to correspond to the inhibitory potency of other snail galactans.     

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.     

Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461

Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [4)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--d-glucose-(1]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP-l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes (gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 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, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Metabolism ofL-sorbose by enzymes fromGluconobacter melanogenus IFO 3293

Summary Studies on the metabolism ofL-sorbose by cell-free extracts fromGluconobacter melanogenus IFO 3293 grown inL-sorbose — containing media confirm by different methodology the observations by Makover et al. that the sequence in this bacterium isL-sorbose L-sorbosone 2-keto-L-gulonic acid L-idonic acid.Dedicated to the memory of Prof. Dr. Konrad Bernhauer. For preceeding paper in this series see Kitamura & Perlman (1975).     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

d-Xylose as inducer of the xylan-degrading enzyme system in the yeast Pullularia pullulans

Summary The specificity of induction of wooddegrading enzymes from Pullularia pullulans was investigated using series of mono-, di- and (14)--trisaccharides or glycanes. A strain of P. pullulans (1740), unable to grow on Avicel or carboxymethyl-cellulose (CMC), uses xylan and steamexploded wood as carbon sources. This strain, thus grown, was evaluated for various enzyme activities. d-Xylose was the nutritional inducer of -xylosidase and -xylanase. d-Glucuronic acid induced activity on CMC and -glucosidase activity was observed regardless of carbon source used. (14)--Xylobiose was not an inducer of -xylanase production, but high levels of this enzyme were obtained with either structural isomers (12) or (13)-. Since synthesis of this enzyme was stimulated by increasing xylose concentration yp to 40 g/l, it is suggested that xylose enters the cells by passive transport and is unable to induce a permease system.Affiliated to the Scientific, Technological and Medical University of Grenoble     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Synthesis of chemically modified sialic acid-containing sialyl-LeX ganglioside analogues recognized by the selectin family

Sialyl Lewis X ganglioside analogues containing 5-acetamido-3,5-dideoxy-l-arabino-2-heptulopyranosylonic acid (C7-Neu5Ac), 5-acetamido-3,5-dideoxy-d-galacto-2-octulopyranosylonic acid (C8-Neu5Ac), and 5-acetamido-3,5-dideoxy-l-glycero-d-galacto-1-2-nonulopyranosylonic acid (8-epi-Neu5Ac) in place ofN-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl 6-O-benzoyl--d-galactopyranoside with the phenyl or methyl 2-thioglycoside derivatives of the respective sialic acids, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid as a promoter in acetonitrile, gave the three required 2-(trimethylsilyl)ethyl (2S)-sialyl-(2 3)--galactopyranosides. These were converted viaO-benzoylation, selective transformation of the 2-(trimethylsilyl)ethyl group to acetyl, and introduction of the methylthio group with methylthiotrimethylsilane into the corresponding glycosyl donors. Glycosylation of 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(1 3)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(1 3)-2,4,6-tri-O-benzyl--d-galactopyranoside with these donors in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) afforded the expected -glycosides, which were converted into the corresponding -trichloroacetimidates, and these, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the required -glycosides. Finally, these were transformed via selective reduction of the azide group, condensation with octadecanoic acid,O-deacylation, and de-esterification into the target compounds in good yields.     

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.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Differential binding characteristics and applications of dGalβ1→3dGalNAc specific lectins

Summary The binding properties of Arachis hypogaea (PNA), Bauhinia vurpurea alba (BPL), Maclura pomifera (MPL) and Sophora japonica (SJL) lectins were studied by quantitative precipitin and precipitin inhibition assays, demonstrating them to be most specific for dGal13dGalNAc residues. Additionally, each lectin had its own binding characteristic such as different binding abilities to dGal14dGlcNAc or dGal13dGlcNAc1linked oligosaccharides, and/or dGalNAc1linked to the Ser or Thr of the protein moiety. These differential binding characteristics can be used for investigating fine differences of the carbohydrate structure of the glycoconjugates, especially those having dGal13dGalNAc residues as terminal non-reducing ends.Abbreviations dGal d-galactopyranose - dMan d-mannopyranose - dGalNAc 2-acetamido-2-deoxy-d-galacto-pyranose - dGlcNAc 2-acetamino-2-deoxy-d-glucopyranose - LFuc L-fucose - NeuNAc N-acetylneuraminic acid - Ser serine - Thr Threonine - RCA Ricinus communis agglutinin - SBA Soy bean agglutinin (Glycine max) - HPA Helix pomatia agglutinin - DBL Dolichos biflorus lectin - GCL Geodia cydonium lectin     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Purification and properties of a novel β-galactosidase or exo-(1 → 4)-β-d-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 4)--linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a -d-galactosidase or exo-(1 4)--d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited -galactosidase action, catalysing the hydrolysis of p-nitrophenyl--d-galactopyranoside and (1 4)- and (1 6)--linked galactobioses. Lactose [-d-galactopyranosyl-(1 4)-d-glucose] was hydrolysed only very slowly and methyl--d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing -d-galactopyranosyl residues were not substrates. A linear (1 4)--linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, -galactonolactone and Cu⁺² were inhibitory. No endo--d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 4)--galactan component of the cell wall.Abbreviations CM carboxymethyl - DEAE diethylaminoethyl - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography We wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the award of a studentship to M.S. Buckeridge, and the Government of São Paulo State, Brazil for granting him leave of absence. We are grateful to Dr. Amanda Heyller (Unilever Research Laboratory, Colworth House, Bedford, UK) for N-terminal sequence determinations, to Dr. Stuart Wilson (Stirling) for preparing gelatin SDS-gels and to Cristina Fanutti (Stirling) for purifying the xyloglucan oligosaccharide.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

A Gln-281 to Arg substitution in α-l-fucosidase is responsible for a common polymorphism detected by isoelectric focusing

A common polymorphism of human -l-fucosidase consists of three phenotypes (Fu 1, Fu 2, and Fu 2-1) assigned by isoelectric focusing. The phenotypes are determined by two codominant alleles (Fu¹ and Fu²). Isozymes with the Fu 2 phenotype have more basic pI than Fu 1, while Fu 2-1 is a mixture of Fu 2 and Fu 1. Recently, a missense mutation (A⁸⁶⁰G) in the -l-fucosidase gene was described that did not affect -l-fucosidase activity. The mutation causes the substitution of Arg (pKa^Guan=12.5) for Gln-281, which has no ionizable side chain. Isoelectric focusing profiles of extracts of COS-1 cells transfected with wild-type or mutant -l-fucosidase cDNAs had phenotypes of Fu 1 and Fu 2, respectively. Next, 20 human lymphoid cell lines were examined for the occurrence of the A⁸⁶⁰G mutation and expression of the Fu 1, Fu 2, and Fu 2-1 phenotypes. Eight lines with Fu 2 were homozygous for the A⁸⁶⁰G mutation; six lines with Fu 1 were homozygous for the normal nucleotide (A⁸⁶⁰); and six lines with Fu 2-1 were heterozygous. Thus, the A⁸⁶⁰G mutation is the molecular basis for the protein phenotypes and the Fu¹ and Fu² alleles. The normal nucleotide (A⁸⁶⁰) is responsible for Fu¹ and the A⁸⁶⁰G mutation for Fu².     

Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins

Glycoproteins (gps) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. They are present on the cell surface and function as receptors in various life processes. Many exist in soluble or gel form and serve as biological lubricants or as barriers against microbial invasion. During the past two decades, eleven mammalian structural units have been used to express the binding domain of applied lectins. They are:F, GalNAc1 3GalNAc;A, GalNAc1 3Gal;T, Gal1 3GalNAc;I, Gal1 3GlcNAc;II, Gal1 4GlcNAc;B, Gal1 3Gal;E, Gal1 4Gal;L, Gal1 4Glc;P, GalNAc1 3Gal;S, GalNAc1 4Gal andTn, GalNAc1 Ser(Thr). ExceptL andP, all of the units can be found in glycoproteins.Tn, which is an important marker for breast/colon cancer and vaccine development, exists only inO-glycans. NaturalTn gp, the simplest mammalianO-glycan, is exclusively expressed in the armadillo salivary gland. Antifreeze gp is composed of repeating units ofT.Pneumococcus type XIV capsular polysaccharide has uniformII disaccharide as carbohydrate side chains. Asialo human ₁-acid gp and asialo fetuin provide multi-antennaryII structures. Human ovarian cyst gps, which belong to the complex type of glycoform, comprise most of the structural units. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding properties of Gal/GalNAc reactive lectins have been classified according to their highest affinity for structural units and their binding profiles are expressed in decreasing order of reactivity. Hence, the binding relationship between glycoproteins and Gal/GalNAc specific lectins can be explored.