Lectin-associated proteins from the seeds of Leguminosae

The seeds of Pisum sativum (pea), Canavalia ensiformis, Vicia faba, Vicia sativa, and Ricinus communis were shown to contain proteins which are associated to the respective lectins (lectin binders). The lectin binders from Pisum sativum and Canavalia ensiformis were studied more closely. Both are single proteins not resembling the variety of membrane glycoproteins found in animals and plants which bind to lectins. The pea lectin binder is a tetrameric glycoprotein composed of identical subunits of the Mr 51 000. Its interaction with the lectin is abolished by acidic buffers or by glucose. The Concanavalin A binder, which does not contain sugar, is composed of one kind of subunit, Mr of 35 000. As in the case of the pea lectin binder, glucose and acid dissociate the lectin-lectin binder complex, but in contrast to the pea lectin binder low NaCl concentrations also cause this effect. During germination and growth, the Concanavalin A binder appears in the roots.     

Purification and characterization of a mitogenic lectin and a lectin-binding protein from Vicia sativa

From the seeds of Vicia sativa, a novel mitogenic lectin was isolated. Purification was carried out by affinity chromatography on Sephadex G-100. The tetrameric lectin is a glycoprotein with a molecular weight of Mr 40 000; it consists of two large beta-subunits (Mr 14 000) and two small alpha-subunits (Mr 6000). The N-terminal sequence of both subunits and their amino acid compositions were determined. The lectin agglutinates human erythrocytes, preferring group B, and erythrocytes from rabbits and horses; no agglutination takes place with sheep erythrocytes. Agglutination is inhibited by mono-, di- and tri-saccharides with the configuration of glucose at the free 4-hydroxyl group. The lectin stimulates mitosis in lymphocytes of mice. From the seeds of the same plant, a protein was isolated which binds to the lectin described above. The lectin binder consists of subunits with a molecular weight of 53 500.     

Isolation and characterization of lactose-binding lectins from the venoms of the snakes Lachesis muta and Dendroaspis jamesonii

Two lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jamesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28,000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26,000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.     

A novel galactose- and arabinose-specific lectin from the sponge Pellina semitubulosa: isolation, characterization and immunobiological properties.

A new lectin from the sponge Pellina semitubulosa is derived which was extracted and purified to homogeneity. The purified lectin is probably a hexamer of polypeptide chains (each M(r) 34,000) which are covalently linked via disulfide linkages; the isoelectric point is 6.1. The lectin displays the following specificities: D-galactose (50% inhibition of hemagglutination at 0.2 mM) = L-arabinose (0.2 mM) greater than D-fucose (1.5 mM) greater than D-glucose (3.0 mM). It precipitates human erythrocytes (A1, A2, A1B, B, and O) with a titer between 2(8) and 2(11) and erythrocytes from sheep and rabbits with a titer between 2(5) and 2(10). The Pellina lectin displays a strong mitogenic effect on spleen lymphocytes from mice. Immunochemical analyses revealed that both murine T- and B-lymphocytes display a capping of the lectin receptors on their cell surfaces after lectin treatment. Murine macrophages were found to endocytose the lectin. Pellina lectin at concentrations between 0.3 and 10.0 micrograms/ml potently enhances interleukin 1 (IL-1) release from mouse peritoneal macrophages and interleukin 2 (IL-2) production in mixed murine lymphocyte cultures.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Purification and Mass Spectrometric Characterization of Sesbania aculeata (Dhaincha) Stem Lectin

A glucose specific lectin (STA) was isolated from Sesbania aculeata stem by using Sephadex G-50 affinity column chromatography. The lectin is a glycoprotein having 29 kDa subunit molecular weight. Two dimensional gel electrophoresis analysis revealed that the lectin existed in two isomeric forms with varied carbohydrate content as analyzed by high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and N-terminal sequence (LDSLSFTYNNFE) analysis of this lectin showed 95% homology with stem lectin SL-I (accession no. AJ585523) from peanut plant. The nucleotide sequence of the lectin (STA) was submitted to the gene bank (accession no. EU263636).     

Isolation and partial characterization of an N-acetylgalactosamine-specific lectin from winter-aconite (Eranthis hyemalis) root tubers.

A lectin was isolated from root tubers of winter aconite (Eranthis hyemalis) by affinity chromatography on fetuin-agarose, and it was partially characterized with respect to its biochemical, physicochemical and carbohydrate-binding properties. The Eranthis hyemalis lectin is a dimeric protein (Mr 62000) composed of two different subunits of Mr 30000 and 32000, held together by disulphide bonds. It is especially rich in asparagine/aspartic acid, glutamine/glutamic acid and leucine, and contains 5% covalently bound carbohydrate. Hapten inhibition assays indicated that the winter-aconite lectin is specific for N-acetylgalactosamine. In addition, the lectin exhibits a pronounced specificity towards blood-group-O erythrocytes. The winter-aconite lectin is the first lectin to be isolated from a species belonging to the plant family Ranunculaceae. It appears to be different from all previously described plant lectins.     

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

In vivo binding partners of the Lens culinaris lectin

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.     

A D-mannose-specific lectin from Gerardia savaglia that inhibits nucleocytoplasmic transport of mRNA

A new lectin has been isolated from the coral Gerardia savaglia by affinity chromatography, using locust gum as an absorbent, and D-mannose as eluant. Final purification was achieved by Bio-Gel P300 gel filtration. The agglutinin is a protein composed of two polypeptide chains with a Mr of 14800; the two subunits are not linked by disulfide bond(s). The isoelectric point is 4.8, the amino acid composition is rich in the acidic amino acids aspartic acid and glutamic acid. The absorption maximum for the protein was at 276 nm; with a molar absorption coefficient of 1.27 X 10(5) M-1 cm-1. The lectin precipitated erythrocytes from humans (A, B and O), sheep, rabbit and carp with a titer between 2(5) and 10(10); the affinity constant for lectin binding to sheep red blood cells was 2.8 X 10(8) M-1 and the number of binding sites, 3.2 X 10(5)/cell. Ca2+ ions are required for full activity; the pH optimum lies in the range between 6 and 11. Inhibition experiments revealed that the lectin is specific for D-mannose. The lectin is mitogenic only for those spleen lymphocytes from mice which had been activated by lipopolysaccharide. An interesting feature of this lectin is its ability to bind to glycoproteins present in nuclei from CV-1 monkey kidney cells. The fluorescein-isothiocyanate-labelled lectin reacted with six polypeptides in the nuclear envelope from rat liver (Mr 190,000, 115,000, 80,000, 62,000, 56,000 and 42,000) and with two polypeptides in the nuclear matrix or pore complex lamina fraction (Mr 190,000 and 62,000). The lectin inhibited the nuclear envelope mRNA translocation system in vitro. It is suggested that this effect is due to an interaction of the lectin with the nuclear glycoproteins gp190 and/or gp62.     

Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

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.     

Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF

The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains. In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography. In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit. The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii. It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains. The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6. The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked. Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24%. Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.     

The isolation of a rat alveolar macrophage lectin

A lectin in rat alveolar macrophage membranes with a high affinity for binding ligands containing L-fucose and N-acetyl-D-glucosamine has been isolated by affinity chromatography on Fuc-BSA-Sepharose (where Fuc is fucosyl and BSA is bovine serum albumin). The lectin was extracted from rat lung homogenates with Triton X-100, absorbed from the extract onto Fuc-BSA-Sepharose in the presence of Ca2+ and eluted by removal of Ca2+. After a second adsorption to and elution from Fuc-BSA-Sepharose, three protein species were detected electrophoretically in fractions that bind Fuc-BSA. One, which was the mannose/N-acetylglucosamine lectin (Mr = 32,000) found earlier in hepatocytes, was removed by adsorption on anti-lectin IgG-Sepharose. Another (Mr = 46,000) was removed by adsorption to Fuc-BSA-Sepharose and elution with galactose. The remaining lectin (Mr = 180,000) bound fucose and N-acetylglucosamine but not galactose. Binding was maximal between pH 6.5 and 9.0 and dependent on Ca2+. Immunocytological analysis with rabbit anti-lectin IgG and fluorescein-labeled goat anti-rabbit IgG revealed the lectin to be in rat alveolar macrophages and nonparenchymal cells of liver. Thus, the lectin appears to be present in macrophages and is likely involved in receptor-mediated endocytosis. It is distinctly different structurally from the hepatocyte lectin with a similar ligand-binding specificity.     

Inhibition of protein synthesis by a toxic lectin from Viscum album L. (mistletoe).

1. The haemagglutinating and toxic lectin from Viscum album L. (mistletoe) inhibits protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 2.6 microgram/ml. This effect is enhanced (ID50 0.21 microgram/ml) if the lectin is reduced with 2-mercaptoethanol. 2. The lectin inhibits protein synthesis also in BL8L cells in culture. Inhibition occurs after a lag time of 3 h. The ID50 is 7 ng/ml, and increases after reduction of the lectin. 3. This and the gross lesions observed in rats poisoned with V. album lectin indicate this is a toxin very similar to ricin.     

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.     

Purification of lectin from fruiting bodies of Lactarius rufus (Scop.: Fr.)Fr. and its carbohydrate specificity

A lectin from fruiting bodies of Lactarius rufus (Scop.: Fr.)Fr. has been purified by affinity chromatography on copolymer of polyvinyl alcohol with a blood group B specific substance. The lectin gives a single band at disk-electrophoresis in acidic (pH 4.3) and alkaline (pH 8.6) buffer systems. Under electrophoresis in 10-20% SDS-PAGE, the lectin consists of identical subunits with molecular weight 17 +/- 1 kDa. Molecular weight of the lectin is 98 kDa according to gel-chromatography on Tojopearl HW-55. It is supposed that the lectin contains six subunits. The lectin is quite enough stable in pH 4.0-10.0, its activity does not depend upon bivalent metal ions. When heating the lectin solution to 65 degrees C it lost more than 85% of its activity. The lectin agglutinates human etrythrocytes without any marked group specificity, it agglutinates 2-4 times worse rabbit erythrocytes, very weakly crucian erythrocytes and does not agglutinate sheep erythrocytes. Mono- and disaccharides are not inhibitors of the lectin activity, while alpha-phenyl-N-acethyl-D-glucosaminopyranosid (0.08 mM) and 4-nitrophenyl-beta-D-glucosamin are the best inhibitors of its activity. Among glycoproteins the best inhibitors of the lectin activity are: group-specific substances from human blood erythrocytes, asialosubmaxillary bovine mucin, human and bovine thyroglobulin and more weak inhibitors are fetuin, transferrin and human Ig G.     

Determination of lectin characteristics by a novel agglutination technique.

A technique generally applicable for the determination of lectin characteristics is described. A sensitive light transmission/scattering method was adapted for the determination of lectin levels and lectin activity. Applying this procedure Geodia cydonium lectin-mediated agglutination was studied in an agglutimeter device using erythrocytes and even T-lymphocytes. In the Geodia lectin/T-lymphocyte system chosen, (i) a lectin concentration as low as 0.57 micrograms/ml could be measured accurately, (ii) the observed cell agglutination velocity constant with a maximal value of 0.75 min-1 was calculated, and (iii) the size of the agglutinates at a given lectin concentration and time period was estimated. The Geodia lectin activity was determined in parallel also in the erythrocyte system. Here, compared to the lectin/T-lymphocyte system the agglutination efficiency of the Geodia lectin-mediated agglutination was more than 10-fold higher and the lowest detectable lectin concentration was 0.06 micrograms/ml. Compared to the hemagglutination assay the lectin/erythrocyte system turns out to be more sensitive and to give much more information on agglutination behavior; this conclusion is supported by additional data using a second lectin isolated from Pellina semitubulosa. The superiority of the agglutination method described here over other known methods must be seen in its accuracy; moreover more lectin characteristics can be determined.     

Purification of lectin from perch (Persa fluviatilis L.) roe specific to cellobiose and study of its characteristics

Two lectins with different carbohydrate specificity were purified from perch (Persa fluviatilis L.) roe (coastal ecological form) by affinity chromatography on ovariomucine H-sepharose from a human ovary cyst. One lectin was eluted by cellobiose and another lectin was eluted by L-fucose. The L-fucose-specific lectin interacted only with L-fucose and its derivatives, but did not interact with cellobiose and salicin. The cellobiose-specific lectin interacted with all the examined carbohydrates, but cellobiose was the best inhibitor. This lectin can be also purified on cellulose as an affinity sorbent. Unlike the L-fucose-specific lectin from perch roe, the cellobiose-specific lectin is less soluble in water-saline solutions. Lectin solubility increases greatly in presence of specific inhibitors, cellobiose, in particular. L-fucose, alpha-methyl-L-fucopyranoside and 4-nitrophenyl-alpha-L-fucopyranoside are equivalent inhibitors for both lectins. According to SDS-PAGE data, the lectins contain two components with molecular weight 12-13 kDa. In solutions, these components form molecules with 50 or 100 kDa (depending on pH). Data obtained from electrophoresis in PAAG in alkaline (pH 8.9) and acidic system (pH 4.3), and SDS-PAGE did not display essential distinctions between these both lectins.     

Aggregation of sponge cells. Function of a lectin in its homologous biological system.

For the first time, the biological role of a lectin in the process of reaggregation of single cells from the same species (marine sponge: Geodia cydonium Jam.) is described. The galactose-specific lectin does not promote aggregation, but prevents the antiaggregation receptor from disaggregating cell clumps. Competition experiments showed that the lectin inactivates the antiaggregation receptor by binding to it, most likely via its terminal galactose residues. The lectin converts reversibly aggregation-deficient cells (carrying functional cell membrane-bound antiaggregation receptor molecules) to aggregation-susceptible cells.