Cell binding and mitogenic properties of the lima bean lectins

Two lectins, a tetramer designated LBL₄ and an octamer LBL₈ designated have been purified from the lima beanPhaseolus lunatus. The tetramer appears to be nonmitogenic for human lymphocytes and is a weak mitogen for bovine cells. The octamer and a chemically cross-linked form of the tetramer are good mitogens. The lima bean lectin binds to only certain sub-populations of human lymphocytes. The primary class which does not bind appears to be a sub-population ofT-lymphocytes. Comparisons of cell binding with other lectins which bind to 2-acetamido-2-deoxy-D-galactose have been carried out. Quantitative analysis of the binding to human erythrocytes is co-operative but binding to lymphocytes is non-co-operative. These results show that there may not be a direct correlation between mitogenic stimulation and cooperative binding to membrane receptors.     

Binding-site specificity of lectins from Bauhinia purpurea alba,Sophora japonica,and Wistaria floribunda

The binding-site specificities of lectins isolated from the seeds of Baihinia purpurea alba, Sophora japonica, and Wistaria floribunda were studied by hemagglutination-inhibition assays utilizing a variety of saccharides as inhibitors. For Bauhinia lectin, 2-acetamido-2-deoxy-d-galactose was found to be the best monosaccharide inhibitor and the free monosaccharide inhibitor was as active as its glycosides. d-Galactose was a weak inhibitor and so were some of its glycosides. Some of the oligosaccharides having a d-galactose nonreducing terminus were good inhibitors, but substitution on the d-galactose or 2-acetamido-2-deoxy-d-galactose residues with other saccharides abolished the inhibitory activity. No specificity for anomeric configuration or linkage position could be demonstrated. The presence of aromatic aglycon groups did not enhance inhibitory activity of the saccharides tested and, in some cases, the inhibitory activity was decreased. In contrast to the results for the Bauhinia lectin, compounds having aromatic aglycon groups were markedly better inhibitors for Sophora and Wistaria lectins than the corresponding compounds without aromatic aglycons. d-Galactose was a weak inhibitor for Sophora and Wistaria lectins, whereas 2-acetamido-d-galactose was a poor inhibitor of Sophora lectin but a good inhibitor of Wistaria lectin. Sophora and Wistaria lectins were somewhat similar in their activity as some of the saccharides having a d-galactose in penultimate position to an l-fucose residue were weak inhibitors. However, Sophora lectin has a binding preference for β anomers, whereas Wistaria lectin did not demonstrate a clear preference for α or β anomers. For some pairs of compounds, the α was a better inhibitor than, the β anomer; in other cases, the reverse was true.     

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.     

Valency-dependent stimulating effects of lima bean lectins on lymphocytes of different species.

Di- and tetravalent lectins purified from lima beans have mitogenic activity towards human, bovine, rabbit, rat and probably mouse lymphocytes; the effect of the mitogen varies for the different species. The mitogenic activity of the 2 lima bean lectins is related to their valency: LIM 124, the component with molecular weight 124 000 and 2 saccharide binding sites, is a weak mitogen; LIM 247, the component with molecular weight 247 000 and four saccharide binding sites, is several times more active. There are indications that the tetravalent LIM 247 exhibits B cell stimulatory activity.     

Developmental changes in the bark lectin of Sophora japonica L.

Lectin is the major protein in the phloem tissue of S. japonica. By immunohistochemistry using anti-seed lectin antibody it was demonstrated that the lectin was localized in the ray and the axial parenchyma. Neither lectin nor other cross-reactive materials were observed in the cambium, sieve tubes and companion cells. The distribution and localization changed in relation to tissue development. Lectin content in the bark changed during the year, the average in summer being about 50% of that in winter. The distribution of lectin in the bark in winter was similar from the innermost (youngest) to the outermost (oldest) region. In contrast, in summer the innermost region hardly contained any lectin, and the outermost region contained less lectin than the middle. Lectin localization in tissues and cells differed also depending on tissue age. In new tissue, produced in the current year, lectip was absent in summer, was located in the cytoplasmic layer between cell wall and vacuole in autumn, and sequestered in the vacuoles in winter. On the other hand, lectin in old tissue (formed in the previous year) was located throughout the year mainly within the vacuoles, with only very small contents in the cytoplasmic layer in autumn. Within the outermost (oldest) region, in which the lectin content was low in summer, the cells which bordered the outer bark never contained any lectin in summer. The intracellular localization in autumn in new tissue, determined by immunogold electron microscopy, was in the lumen of the endoplasmic reticulum and vesicles, with gold particles hardly present in the cytoplasm. From these findings we conclude that lectin is synthesized on the endoplasmic reticulum and most vigorously in the new tissue in autumn, and that it is mainly consumed in the outermost bark regions, where dilatation occurs and-or where cork cambium is differentiated.Abbreviations ELISA enzyme-linked immunosorbent assay - ER endoplasmic reticulum - kDa kilodalton Retired. Anatomical terms in this paper are used according to Multilingual glossary of terms used in wood anatomy edited by the Committee on Nomenclature, International Association of Wood Anatomists; reprints may be obtained from the Office of the Secretary-Treasurer, Universitätsstrasse 2, CH-8092 Zürich 6, Switzerland.     

Isolation and characterization of a cDNA clone encoding the lima bean lectin

The complete amino acid sequence of the lima bean (Phaseolus lunatus) lectin was deduced from the nucleotide sequence of a cDNA clone. The lectin appears to be synthesized as a prepeptide consisting of a signal sequence of 21 residues and a mature protein of 241 amino acids. Comparison of the lima bean lectin sequence to the sequences of other leguminous seed lectins indicates regions of extensive homology. Northern blot analysis showed absence of lectin mRNA in the leaves, roots, or stems of 16-day-old lima bean plants.     

Oxidation and chemical modification of lung beta-galactoside-specific lectin.

Galaptins are small, soluble, lectins with a specificity for beta-galactose residues. Many galaptins are inactivated by atmospheric oxygen and are protected by disulphide-reducing reagents. We find that each subunit of rat lung galaptin contains one residue of tryptophan and six of cysteine. Oxygen inactivates rat lung galaptin by oxidation of the cysteine residues. During oxidation, the normal dimeric structure is maintained and all disulphide bonds are formed within individual subunits. Exogenous thiols protect against inactivation, but oxidized thiols accelerate inactivation. Human lung fibroblast galaptin is almost completely inactivated within 1 h in tissue culture medium at 37 degrees C. Alkylation of native rat lung galaptin with iodoacetate or ethyleneimine causes substantial loss of activity. The dimeric galaptin structure is maintained. In contrast, alkylation with iodoacetamide yields carboxamidomethyl-galaptin, which is fully active and stable to atmospheric oxygen in the absence of disulphide-reducing reagents. This derivative is very useful for studies of galaptin properties and function.     

Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity.

1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.     

Alteration of the carbohydrate-binding specificity of the Bauhinia purpurea lectin through the preparation of a chimeric lectin.

A chimeric lectin gene was constructed by using a cDNA clone coding the Bauhinia purpurea lectin (BPA) in which a part of the metal-binding region was replaced by the corresponding region of the mannose-binding Lens culinaris lectin (LCA). The chimeric lectin expressed in Escherichia coli was found to bind alpha mannosyl-bovine serum albumin (BSA) and this binding was inhibited by mannose.     

Antigenic and calcium binding properties of a Peptide containing the essential cysteine in lima bean lectin

Polyclonal antisera were raised against a peptide containing the cysteine residue required for carbohydrate binding activity in the lima bean lectin. The antisera were tested for cross-reactivity with (a) synthetic peptide analogs to the essential cysteine containing peptide, (b) proteolytic digests of related lectins, (c) native lectins. The antisera were specifically inhibited from binding to a peptide conjugate by free synthetic peptides. The degree of inhibition by lectin digests correlated approximately along evolutionary relationships and the degree of sequence conservation. One antiserum was found to cross-react with certain lectins in the native state. In a second set of experiments, the calcium binding properties of the synthetic peptides were investigated using metal ion-chelate chromatography and UV-difference spectroscopy. The nonapeptide and undecapeptide bound to a Ca²⁺ iminodiacetic acid agarose column and were eluted with EDTA. Ultraviolet difference spectral titrations with Ca²⁺ performed on the synthetic undecapeptide and a related favin derived peptide resulted in dissociation constants of approximately 6 × 10³ per molar.     

Preliminary X-ray diffraction analysis of crystalline lectins from the seeds and leaves of Sophora japonica

The Japanese Pogada Tree (Sophora japonica) contains at least four distinct lectins distributed among seeds, bark, and leaf tissues of the plant. All of these lectins are N-acetylgalactosamine-specific, have molecular weights of about 130,000, are glycoproteins and possess substantial sequence homology. However, they are products of distinct genes. We have crystallized Sophora lectins from bark, seeds, and two different lectins from leaves. Multiple crystal forms of each variety have been obtained by vapor diffusion with polyethylene glycol 4,000 solutions, and five of the crystal forms have been characterized by X-ray diffraction. The data demonstrates that at least in the case of leaf lectin II, the tetrameric molecule contains at least one exact dyad axis. Several of the crystals appear suitable for high resolution structure analysis.     

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.     

Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability

5'-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5'-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5'-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5'-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5'-nucleotidase is concanavalin A.     

Primary structure of an N-glycosidic carbohydrate unit derived from Sophora japonica lectin

The lectin isolated from Sophora japonica seeds is a glycoprotein which binds oligosaccharides with non-reducing terminal Gal beta(1----3/4)GlcNac beta 1----units. The carbohydrate moiety of the lectin is composed of fucose, xylose, mannose and N-acetylglucosamine. The major glycopeptide of the lectin, prepared by pronase digestion, was derivatized with fluorescein isothiocyanate, purified by PAGE and examined by exoglycosidase digestion as well as purified by gel filtration through Bio-Gel P6-DG and investigated by methylation analysis and 400-MHz 1H-NMR spectroscopy. The primary structure of the glycopeptide was established to be as follows. (Formula: see text). Structures similar to this containing a (beta 1-2)xylosyl substituent on the core beta-mannosyl residue and an inner core (alpha 1-3)fucosyl substituent seem to occur frequently in plant glycoproteins.     

Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean, Phaseolus vulgaris

8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins.     

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica)

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al.. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.     

Contrasting consequences of plant domestication for the chemical defenses of leaves and seeds in lima bean plants

Plant domestication is assumed to result in reduced levels of defensive compounds in crops, because this makes the plants more suitable for consumption by humans and livestock. We argue that this should mainly be reflected in the concentrations of defense compounds in the plant parts that are used for consumption and not necessarily for other parts of crop plants. We tested this hypothesis for domesticated lima bean (Phaseolus lunatus), by comparing its chemical defenses against a leaf herbivore, the beet armyworm (Spodoptera exigua), and a seed predator, the beetle Zabrotes subfasciatus. For seeds and leaves we determined the concentrations of cyanogenic glycosides (CNGs) in cultivated varieties and wild populations and evaluated the preference and performance of the herbivores when exposed to leaves and seeds from wild and cultivated plants. Concentrations of CNGs were significantly different between wild and cultivated plants. In the leaves the concentration of CNGs in the cultivated varieties were more than double that of the wild leaves. In contrast, seeds from cultivated plants had up to 20 times lower CNG concentration compared to seeds from the wild populations. Insect preference and performance do not parallel the chemical data. Larvae of S. exigua preferred wild leaves but had higher survival on cultivated leaves. The beetles, however, strongly preferred seeds from cultivated plants and females developed more quickly on these seeds. We conclude that domestication of P. lunatus has altered the concentration of CNGs in both the seeds and the leaves in opposite directions. This results in differential effects on the herbivores that attack these two plant structures. The contrasting effect of domestication on different plant tissues can be explained by the fact that bean plants have been specifically selected for human consumption of the seeds. Tissue-specific effects of plant domestication on plant defenses can be expected for other crops as well.     

Role of bean lectins inRhizobium phaseoli-Phaseolus vulgaris interactions. Some properties of lectins from two bean cultivars

Lectins from twoPhaseolus vulgaris L. cultivars were isolated and purified by salt fractionation, affinity chromatography and gel permeation chromatography. The cultivars used were: alubia, with a low-nodulating ability, and Bat 76 with a good symbiotic aptitude. Differences in properties of the two lectins were noted: alubia lectin gave only one peak with haemagglutinating activity following gel permeation chromatography while Bat 76 yielded two active peaks, although both lectins had several bands of about 30 kDa following gel electrophoresis, Bat 76 lecting had three bands of about 50 kDa which were not present in alubia and red kidney bean lectins. Peptide-mapping, by limited proteolysis and two dimensional gel electrophoresis, also showed differences between the lectins which are therefore judged to be different.