A comparison of the carbohydrate binding properties of twoDolichos biflorus lectins

The carbohydrate binding properties of theDolichos biflorus seed lectin and DB58, a vegetative tissue lectin from this plant, were compared using two types of solid phase assays. Both lectins bind to hog blood group A + H substance covalently coupled to Sepharose 4B and this binding can be inhibited with free blood group A + H substance. However, the binding of the seed lectin is inhibited byD-GalNAc whereas DB58 binding was not inhbited by any monosaccharide tested, thus suggesting that its carbohydrate combining site may be more extensive than that of the seed lectin. The activities of these two lectins also differ from one another in ability to recognize blood group A + H substance adsorbed on to plastic and in the effects of salt and urea on their carbohydrate binding activities. Neither lectin showed glycosidase activity with p-nitrophenyl -D-GalNAc or p-nitrophenyl -D-GalNAc.     

A lectin with antifungal and mitogenic activities from red cluster pepper (<Emphasis Type="Italic">Capsicum frutescens</Emphasis>) seeds

A monomeric mannose/glucose-binding lectin, with a molecular mass of 29.5 kDa and an N-terminal sequence GQRELKL showing resemblance to that of the lectin-like oxidized low-density lipoprotein receptor from the rabbit, has been isolated from the seeds of red cluster pepper Capsium frutescens L. var. fasciculatum. The protocol involved anion exchange chromatography on diethylamino ethanol-cellulose and Q-Sepharose and fast protein liquid chromatography on Mono Q. Its hemagglutinating activity toward rabbit erythrocytes was inhibited by d-mannose and glucose, specifically. The activity was stable from 0 to 40°C, reached a maximum at pH 7 and 8, and was potentiated by Ca²⁺ and Mn²⁺ ions. The lectin showed strong mitogenic activity toward spleen cells isolated from BALB/c mice. The mitogenic activity, which reached a peak at a lectin concentration of 0.27 μM, was inhibited specifically by d(+)-mannose. The lectin was capable of inhibiting the germination of Aspergillus flavus and Fusarium moniliforme spores and hyphal growth in the two fungi.     

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study

The X-ray crystal structure of lentil lectin in complex with -d-glucopyranose has been determined by molecular replacement and refined to anR-value of 0.20 at 3.0 Å resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I fromLathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the -d-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin.Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)--d-mannopyranoside and Me-3-O-(m-nitro-benzyl)--d-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 and Asn125, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 and Trp128.RL and FC are joint first authors.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Regulation of mesophyll photosynthesis in intact wheat leaves by cytoplasmic phosphate concentrations

The effect of D-(+)-mannose, inorganic phosphate (Pi) and mannose-6-phosphate on net mesophyll CO₂ assimilation rate (A) and stomatal conductance (g_s) of wheat (Triticum aestivum L.) leaves was studied. The compounds were supplied through the transpiration stream of detached leaves from plants grown in sand in growth cabinets or glasshouses, with different concentrations of Pi (0.25, 1.0 and 4.0 mM) supplied during growth. In all cases, 10 mM D-(+)mannose caused 40–60% reduction of A within 30 min, though the time courses differed for flag leaves and the sixth leaf on the mainstem of glasshouse- and cabinet-grown plants. D-(+)Mannose had a similar effect on A in leaves having a fourfold range in total phosphate content. Effects of D-(+)mannose in reducing g_s were always slower than on A. When the CO₂ concentration in the leaf chamber was adjusted to maintain intercellular CO₂ concentration (C_i) constant as A declined after mannose supply, g_s still declined indicating that stomatal closure was not caused by changing C_i. Supplying mannose-6-phosphate at 10 and 1 mM and Pi at 5 and 10 mM concentrations caused rapid reductions in g_s and also direct reductions in A. The observed effects of mannose and Pi on assimilation are consistent with the proposed regulatory role of cytoplasmic Pi in determining mesophyll carbon assimilation that has been derived previously using leaf discs, protoplasts and chloroplasts.Abbreviations and symbols A net mesophyll CO₂-assimilation rate - C_a, C_i external (assimilation-chamber) and intercellular CO₂ concentration, respectively - g_s stomatal conductance - Man6P mannose-6-phosphate - Pi orthophosphate     

Purification and characterization of mannose/glucose-specific lectin from seeds of <Emphasis Type="Italic">Trigonella foenumgraecum</Emphasis>

A lectin present in seeds of Trigonella foenumgraecum was isolated and purified by acid precipitation, salt fractionation, and affinity chromatography on mannan cross-linked agarose. SDS-PAGE revealed a single band corresponding to a molecular weight of 27,350 daltons. The lectin agglutinated trypsin-treated rat erythrocytes. Sugar specificity as determined by hemagglutination inhibition assay indicated that the lectin belongs to a glucose/mannose-specific group. The reaction of the lectin with glycoprotein was affected by pH changes. The carbohydrate binding specificity of the lectin was investigated by turbidity and activity measurements. As the lectin belongs to the Leguminoceae family, the specificity of the lectin for glucose/mannose renders it a valuable tool for Rhizobium-legume symbiosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 52–57.     

Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.     

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.     

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.     

Mannose-Specific Lectin Activity of Parasporal Proteins from a Lepidoptera-Specific <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori. Received: 21 February 2002 / Accepted: 28 March 2002     

Use of antisera to localize callose,xylan and arabinogalactan in the cell-plate,primary and secondary walls of plant cells

Antibodies to cellobiose, L-arabinopyranose, L-arabinofuranose, D-galactose, oligosaccharides containing 14 xylose, oligosaccharides containing 14 glucose, and oligosaccharides containing 13 glucose have been raised in rabbits. The antisera have been characterized to show the specificity of binding to particular polysaccharides. They have been used for immunocytology using the electron microscope to locate the polymers in dividing and differentiating cells of bean (Phaseolus vulgaris L.) root, bean callus tissue and cells of Zinnia elegans L. in vitro. Arabinogalactans have been shown to be present in the cell-plate and primary walls but not in secondary thickening. Xylan as distinct from xyloglucan was found in the primary walls but not in the cell-plate. It was present in large amounts in the secondary thickening. Callose was found in the cell plate and also in the young growing wall. In the wall it was specifically located at the plasmodesmata. The use of the antibody against L-arabinofuranose enabled a specific organelle to be detected which was membranous and which occurred within the cytoplasm and also within the vacuole of the cells. Membranes carrying polymers containing L-arabinofuranose were also found in layers just under the plasmamembrane.Abbreviations L-Araf L-arabinofuranose - L-Arap L-arabinopyranose - BSA bovine serum albumin - Gal galactose - D-Galp D-galactopyranose - Glc glucose - Xyl xylose     

Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)

For the first time a sugar receptor (lectin) has been localized by electron microscopy in an invertebrate. The peritrophic membrane of the blowfly larva, Calliphora erythrocephala, is shown here to express lectins with high specificity for mannose. The lectin is restricted to the lumen side of the peritrophic membrane. The surface of the midgut epithelium is devoid of mannose-specific lectins. It is suggested that the midgut epithelium has lost these lectins during the course of evolution in favour of the peritrophic membrane which is secreted by specialized cells only at the beginning of the midgut.Peritrophic membranes and the midgut epithelium lack lectins specific for galactose. The lumen side of the peritrophic membrane of the larvae has mannose and/or glucose residues, and it is densely packed with two species of bacteria, Proteus vulgaris and P. morganii. These also have mannose-specific lectins as well as mannose residues on their pili. The existence of mannose-specific receptors and mannose residues on both, peritrophic membranes and bacteria, leads to the assumption of mutual adherence between the two surfaces.     

The lectin nature of α-galactosidases from Vicia faba seeds

α-Galactosidase from Vicia faba seeds has been resolved into three molecular forms, I, II¹ and II², respectively. Enzyme I is a tetramer (M_r 160 000) consisting of identical sub-units (M_r 44000 ± 2000). All three forms display lectin activity with glucose/mannose specificity. Enzyme I has been further studied with respect to its lectin specificity and various factors affecting this property. The results indicate that the catalytic and the lectin sites reside in the same protein molecule. The results presented are unique in that the enzyme activity is specific for galactose and its lectin activity is specific for glucose/mannose.     

Purification and characterization of a D‐mannose specific lectin from the green marine alga,Bryopsis plumosa

A d ‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d ‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l ‐fucose (8 mM). d ‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.     

Enolization and racemization reactions of glucose and fructose on heating with amino-acid enantiomers and the formation of melanoidins as a result of the Maillard reaction

This study investigated the enolization and racemization reactions of glucose and fructose on heating with amino acid enantiomers and the formation of melanoidins as a result of the Maillard reaction. The study measured reducing sugars and L- and D- amino acids using HPLC as an index for the amount of enolization of the sugars and isomerization of the amino acids. Additionally, the absorption of melanoidins was measured at different wavelengths (420, 450, 470, 490 nm); the UV–Vis spectra and the extinction coefficient were determined for the formation of melanoidins. Melanoidins were, rather arbitrarily, defined by a high-molecular-weight (HMW) if it was above a lower limit of 12.4 kDa, which was the nominal cut-off value in the dialysis system used. A remarkable enolization reaction of the sugars was observed in the course of the Maillard reaction. Especially, in the Fru/D-Asn model system, the degree of sugar enolization was more than in the other model systems. All of the FDAA (1-fluoro-2, 4-dinitrophenyl-5-L-alanine amide) amino acids were separated by TLC. The racemization of the amino acids was higher in the fructose-amino acids systems. Isomer formation was the highest in the Fru/D-Asn system. The L- and D- isomers showed different absorptions in the UV–Vis spectra, although these had similar shapes. The absorption of the melanoidins formed from glucose was higher than that formed from fructose. In particular, the sugar–asparagine system showed different characteristics according to the L- and D-isomers. The differences in the extinction coefficients of the melanoidins was significant (P < 0.05), except for the sugar–lysine system.     

d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Cytochemical identification of arabinogalactan protein in the outer epidermal wall of maize coleoptiles

Artificial carbohydrate antigen (Yariv reagent), fluorescence-labeled -l-fucose-binding lectin, and -D-galactose-binding lectin were used to localize arabinogalactan protein in sections of maize (Zea mays L.) coleoptiles. All three probes bind to cell walls of vascular tissue and the outer epidermis. Intense staining is obtained at the outer and inner faces of the growth-controlling outer epidermal wall. At the inner face of this wall the auxin-inducible osmiophilic particles, hitherto observed only by electron microscope (Kutschera et al. 1987, Planta 170, 168–180), are strongly stained by all three probes and can therefore be identified as deposits of arabinogalactan protein. It is proposed that this proteoglycan acts as an epidermal wallloosening factor in auxin-mediated coleoptile growth.Abbreviation AGP arabinogalactan protein I thank Dr. R. Bergfeld for the electron micrograph of Fig. 13. This work was supported by the Deutsche Forschungsgemeinschaft.     

Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe

A photoreactive -D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an -D-glucose moiety; (ii) the digoxigenin tag; (iii) the photoreactive cross-linker; and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe is activated and cross-linked to the CBPs after being treated by several flashes.Using this method we have identified a new -D-glucose CBP ofM _r=33000, termed CBP33, in the nuclei of rats exposed to transient immobilization stress. Monoclonal antibodies were raised against the partially purified protein and subsequently used to enrich CBP33. It was purified (>2400-fold) to apparent homogeneity from a 0.6M nuclear salt extract by two subsequent affinity chromatography steps (antibody-affinity as well as -D-glucose affinity column).Abbreviations BSA bovine serum albumin - CBP carbohydrate binding protein - DIG digoxigenin - Gal galactose - Glc glucose - Lys lysine - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate.     

The impact of supplemental carbon sources on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> growth,chlorophyll content and anthocyanin accumulation

This research explores the impacts of a broad range of supplemental carbon sources on growth and development of Arabidopsis thaliana. Parameters measured include dark-germinated hypocotyl length, light-germinated root growth, rosette growth, chlorophyll concentration and anthocyanin content. Treatment sugars include sucrose, maltose, d-glucose, d-fructose, l-arabinose, l-fucose, d-galactose, d-mannose, l-rhamnose and d-xylose each supplied at 4, 20 or 100 mM. This comparison of the effect of different carbon sources on multiple parameters and under identical conditions showed that every carbon source had unique qualitative and quantitative effects on Arabidopsis growth and development. Root growth was particularly sensitive to supplemental carbon source. Growth on 100 mM sucrose, maltose, glucose or xylose stimulated root growth by ~100%. Growth on arabinose, fucose, galactose, mannose or rhamnose inhibited root growth by 50% or more. Several sugars that strongly inhibited root growth had either no effect (galactose and fucose) or a positive effect (arabinose) on hypocotyl elongation and rosette growth. Rhamnose was the only carbon source that inhibited hypocotyl elongation across all concentrations. Sucrose, maltose, glucose, fructose, arabinose or xylose stimulated rosette growth by ~50%. Chlorophyll content was strongly reduced by mannose while sucrose, glucose, galactose and rhamnose caused smaller reductions. Anthocyanin accumulation was strongly induced by both galactose and mannose. Only mannose impacted all parameters across all concentrations. Based on these data it can be concluded that the effect of each carbon source on Arabidopsis growth and development is specific in terms of both magnitude and the parameters impacted.     

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