Electron-microscopic analysis of ground elder (Aegopodium podagraria L.) lectin: evidence for a new type of supra-molecular protein structure

The lectin of ground elder (Aegopodium podagraria L.) was investigated electron-microscopically after negative staining with uranyl salts. Affinity-purified preparations of this glycoprotein were highly heteromorphous as they contained small particles approximately 4.6 nm in diameter and very large particles of different shapes. Among the latter, circular and helicoidal structures were the most regular in appearance. The circles were 9.3 nm in diameter, whereas the helices were 9 nm or 20 nm in diameter and up to 60 nm in length. After photographic enhancement, pictures of the molecules indicated that both the larger structures and the small particles could be obtained in pure forms by gel filtration of the lectin on Sepharose 4B. Since the former were the only constituents of the excluded fraction (M_r>5000000), whereas they were totally absent in the fraction eluting with an apparent molecular weight of about 500000, these supra-molecular structures revealed by the electron microscope cannot be artefacts generated during preparation of the lectin for electron-microscopic observation.Abbreviations APA Aegopodium podagraria agglutinin - EM electron microscopy - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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).     

Detection and characterization of a lectin from non-seed tissue ofPhaseolus vulgaris

The distribution of lectin in various tissues ofPhaseolus vulgaris L. (cv. red) has been investigated using a sensitive solid-phase enzyme immunoassay. Roots, leaves and stems from 3- to 4-week-old plants were screened for their lectin content; low levels could be detected in all organs, with a relative distribution of 37% in roots, 20% in leaves and 43% in stems. The lectin from stemsleaves and roots was then isolated from 5- to 6-week-old plants using extraction, salt fractionation and affinity chromatography on immobilized porcine thyroglobulin. A comparative study of the seed lectin and the lectin isolated from 5- to 6-week-old plants was made using hemagglutination, inhibition of hemagglutination, immunodiffusion, polyacrylamide and agarose electrophoresis. The results showed that lectin isolated from the different tissues was immunologically identical and exhibited the same subunit structure and similar isolectin composition as the seed lectin.Abbreviations EDTA ethylenediaminetetraacetic acid - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae     

A lectin from Bryonia dioica root stocks

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.Abbreviations BDA Bryonia dioica agglutinin - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Isolation and characterization of glycoprotein lectins from the bark of three species of elder,Sambucus ebulus,S. nigra and S. racemosa

Lectins have been isolated from the bark of three members of the family Caprifoliaceae, Sambucus nigra (elder), S. racemosa (red-berried elder) and S. ebulus (dwarf elder), by affinity chromatography on fetuin-agarose, ion-exchange and gel-filtration chromatography. They are all glycoproteins of M _r 140 000 made up of at least four subunits. The lectin have similar but not identical amino-acid compositions and the carbohydrate content varies between 12% and 19% (w/w), the main sugars being (N-acetyl)glucosamine, mannose, fucose and xylose. Inhibition studies of hemagglutination with various mono- and oligosaccharides have shown that N-acetylgalactosamine and galactose together with galactose-containing oligosaccharides are the most effective inhibitors. There are some differences in specificity, in particular S. ebulus agglutinin is inhibited to the same degree by galactosamine, N-acetylgalactosamine and by galactose.Abbreviations PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SEA S. ebulus agglutinin - SNA S. nigra agglutinin - SRA S. racemosa agglutinin     

Indication of transthylakoid proton-fluxes in Aegopodium podagraria L. by light-induced changes of plasmalemma potential,chlorophyll fluorescence and light-scattering

The time course of the responses of chlorophyll fluorescence in leaves of Aegopodium podagraria to changes in irradiance does not necessarily show the time constant of thylakoid energization at energy fluence rates below 10–25 W·m^-2. In addition, other measures of thylakoid energization, such as lightscattering at 532 nm and the responses to saturating flashes, show that the related component disappears from these signals at low fluence rates, but not necessarily all together at the same fluence rate. However, this time constant still appears in the light-induced responses of the plasmalemma potential. This implies that the effect on the electrogenic proton pump in the plasmalemma is the most sensitive indicator of proton fluxes into the inner thylakoid space. These results are a further indication that energy-quenching is coupled ther indication that energy-quenching is coupled to transthylakoid proton fluxes via an intermediate, which is not active in Aegopodium podagraria at low irradiances.Abbreviations and symbols _i time constant - F chlorophyll fluorescence - I constant component of irradiance - I _v variable component of irradiance - S light-scattering - q _E high-energy state quenching of chlorophyll fluorescence - T transmittance at 532 nm - V plasmalemma potential     

Isolation and partial characterization of a calcium-dependent lectin (chiletin) from the haemolymph of the flat oyster, Ostrea chilensis

A calcium-dependent lectin (chiletin) was isolated from oyster haemolymph by mannose elution from Sepharose CL-6B followed by anion exchange chromatography. Chiletin was predominantly composed of 12 and 24 kDa bands when examined with SDS-PAGE under reducing and non-reducing conditions, respectively. Larger molecular weight bands of 36 and 50 kDa were also variably present under reducing conditions. The NH2-terminal sequence of the 24 kDa band was determined and was not homologous to any known protein from the databases searched. Isolated chiletin was composed of multiple isomers approximately 12 kDa in size and ranging in pI from 5.2 to 6.0. Rabbit antiserum was raised to a synthetic peptide coupled to keyhole limpet hemocyanin and the size of the chiletin subunits was confirmed by Western blot. Two and five different conformational aggregates of chiletin were resolved in oyster haemolymph using size exclusion chromatography in 8 M urea and PBS, respectively. The largest aggregate obtained from size exclusion in 8 M urea was estimated to be greater than 640 kDa. The ability of whole haemolymph and isolated chiletin to agglutinate sheep red blood cells was inhibited by galactose and mannose. Chiletin was identified by immunohistochemistry to be most consistently present in the auricle, followed by the digestive gland, however staining was seen sporadically in haemocytes, gastrointestinal epithelium and interstitial connective tissue cells.     

Differences in properties of peanut seed lectin and purified galactose- and mannose-binding lectins from nodules of peanut

Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal galactose - Gle glucose - GNL galactose-binding nodule lectin - Fru fructose - MNL mannosebinding nodule lectin - M _r rerative molecular mass - PBS phosphate-buffered saline - PSL peanut seed lectin - SDS sodium dodecyl sulphate - Sorb sorbitol     

Developmental changes and tissue distribution of lectin in Tulipa

A sensitive enzyme-immunoassay was developed to quantify the tulip lectin and used to follow its distribution during the life cycle of tulips cv. Attila.The tulip lectin is predominantly located in the bulbs. At planting time the absolute lectin concentration is approximately the same in all bulb scales. However, as the shoot grows and the plant turns on to flowering, the lectin concentration rapidly decreases, first in the inner bulb scales but later also in the outer bulb scale. Soon after flowering the lectin rapidly accumulates in the new daughter bulbs.Lectin levels in leaves, stems and flowers are very low. The lectin in these tissues is already present before the sprout emerges. During the first two weeks after planting, there is a small increase in lectin concentration, followed by a rapid decrease as the plant turns on to flowering. By flowering time all the lectin has disappeared from the aerial parts.Abbreviations DW dry weight - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - PBSN phosphate-buffered saline containing 0.02% sodium azide - PBST phosphate-buffered saline containing 0.02% sodium azide and 0.05% Tween 20 - TL tulip lectin - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A new lectin from tulip (Tulipa) bulbs

A lectin was isolated from tulip (Tulipa) bulbs by affinity chromatography on fetuin-agarose and partially characterized. The tulip lectin is a tetrameric protein composed of four identical subunits of M_r 28 000, which are not held together by disulphide bonds. It is not glycosylated and has an amino-acid composition typified by a high content of asparagine-aspartic acid, leucine, glycine and serine. Tulip lectin agglutinates human red blood cells, but has a much higher specific activity with rabbit erythrocytes. In hapten-inhibition assays with the latter type of red blood cell the lectin exhibits a complex specificity, whereas its agglutination with human erythrocytes is readily inhibited by N-acetylgalactosamine, lactose, fucose and galactose.Abbreviations DEAE diethylaminoethyl - PBS phosphate-buffered saline - TL Tulipa lectin - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Developmental changes and tissue distribution of lectin inGalanthus nivalis L. andNarcissus cv. Carlton

A sensitive immunosorbent assay was developed to quantify the lectin in different tissues ofGalanthus nivalis (snowdrop) andNarcissus cv. Carlton (daffodil) and follow the distribution of the lectin during the life cycle of the plants. The lectin in snowdrops and daffodils occurs in almost all plant tissues. Moreover, in many tissues the lectin is the most prominent protein. High lectin concentrations are found in the bulb where the lectin accounts for up to 15% of the total protein during the resting period. However, as the shoot grows and the plant turns on to flowering the lectin content rapidly decreases. Soon after flowering the lectin accumulates in the new bulb units. Whereas in daffodil the lectin concentration in the aerial plant parts is about one order of magnitude lower than in the bulb, lectin concentrations in the upper parts of snowdrop are similar to those in the bulb. The lectin in the former tissues is already present before the sprout emerges. As the shoot starts to grow lectin concentrations in leaves, stems and flower parts gradually decrease so that at flowering time virtually all lectin has disappeared from the aerial parts. The highest lectin concentrations are found in the ovary and increase, initially, as the sprout emerges from the bulb. This work was supported in part by grants from the ‘Nationale Bank’ and the National Fund for Scientific Research (Belgium). W.J.P. is a Senior Research Associate and E.J.M.V.D. Research Assistant of this fund.     

Isolation and characterization of a novel lectin from the mushroom Armillaria luteo-virens

From the dried fruiting bodies of the mushroom Armillaria luteo-virens, a dimeric lectin with a molecular mass of 29.4 kDa has been isolated. The purification procedure involved (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin could not be inhibited by simple sugars but was inhibited by the polysaccharide inulin. The activity was stable up to 70 degrees C but was acid- and alkali-labile. Salts including FeCl(3), AlCl(3), and ZnCl(2) inhibited the activity whereas MgCl(2), MnCl(2), and CaCl(2) did not. The lectin stimulated mitogenic response of mouse splenocytes with the maximal response achieved by 1microM lectin. Proliferation of tumor cells including MBL2 cells, HeLa cells, and L1210 cells was inhibited by the lectin with an IC(50) of 2.5, 5, and 10 microM, respectively. However, proliferation of HepG2 cells was not affected. The novel aspects of the isolated lectin include a novel N-terminal sequence, fair thermostability, acid stability, and alkali stability, together with potent mitogenic activity toward spleen cells and antiproliferative activity toward tumor cells.     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Isolation of a novel N-acetylglucosamine-specific lectin from fresh sclerotia of the edible mushroom Pleurotus tuber-regium

An N-acetylglucosamine-binding lectin with a molecular mass of 32kDa was isolated from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Its N-terminal sequence exhibited some similarity to that of Agaricus bisporus lectin. The isolation procedure was simple, involving (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on N-acetyl-D-glucosamine-agarose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin exhibited hemagglutinating activity toward trypsinized rabbit erythrocytes but not toward untrypsinized rabbit erythrocytes.