Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Purification and characterization of an α-d-galactosyl-binding lectin from Artocarpus lakoocha seeds

A lectin from Artocarpus lakoocha seeds has been purified by affinity chromatography on a melibiose-agarose column. The homogeneity of the purified lectin was tested by several criteria, viz., poly(acrylamide)-gel electrophoresis, ultracentrifugal analysis, and gel filtration. The molecular weight of the lectin was estimated to be ∼70,000 as determined by Sephadex gel filtration. SDS-poly-(acrylamide)-gel electrophoresis gave a single component of molecular weight 18,000, suggesting that the lectin is a tetramer composed of four apparently identical subunits. The lectin agglutinated human erythrocytes, regardless of blood group. Artocarpus lakoocha lectin is a glycoprotein, and contains 11.7% of carbohydrates, in which d-xylose (6%) is the main sugar, with smaller proportions of d-galactose, d-glucose, d-mannose, N-acetyl-d-glucosamine, and N-acetyl-d-mannosamine. Amino acid analysis of the lectin revealed a high content of acidic and hydroxylic amino acids, a relatively low proportion of basic amino acids, and a trace of cysteine and methionine. In hapten-inhibition assays with simple sugars, glycosides of α-d-galactopyranose and N-acetyl-d-galactosamine were potent inhibitors of the purified lectin.     

Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (M_r) 15,000 which is posttranslationally converted into the authentic lectin polypeptide of M_r 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [³H]leucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organellar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase. Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 millimolar Mg-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M_r 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing.     

Isolation and Partial Characterization of a New Lectin from Seeds of the Greater Celandine (Chelidonium majus)

Seeds of the greater celandine (Chelidonium majus L.) contain a lectin which could be isolated using a combination of affinity chromatography on chitin and ion exchange chromatography on sulphopropyl-Sephadex. The purified lectin was partially characterized with respect to its biochemical and physicochemical properties. It is a small dimeric protein composed of two different subunits of M_r 9,500 and 11,500, respectively. Its amino acid composition is typified by high contents of glycine and cysteine. No covalently bound carbohydrate could be detected. Hapten inhibition experiments indicated that the lectin exhibits specificity towards oligomers of N-acetylglucosamine, the potency of inhibition increasing with chain length up to four residues. The greater celandine lectin is the first lectin to be isolated from a species belonging to the plant family Papaveraceae (poppy family). Although it represents a new type of plant lectin, resemblances to phytohemaglutinins from diverse taxonomic origin are obvious.     

Purification,characterization of mosquito larvicidal lectin from Annona muricata and its eco-toxic effect on non-target organism

The present study investigates the purification and characterization of mosquito larvicidal lectin from the seed kernel extract of Annona muricata and toxic effects on non-target organism Chironomus costatus. Soursop lectin was purified by anion-exchange column chromatography using DEAE-cellulose with approximately molecular mass of 260 kDa and with seven distinct subunits (16, 18, 21, 22, 24, 73 and 95 kDa). Soursop lectin highest Hemagglutination (HA) titer value of 128 was recorded against hen indicator RBC type with the influence of different divalent cations such as Ca²⁺, Ba²⁺ and Mn²⁺. The lectin mediated HA activity was highly inhibited by monosaccharides of glucose and mannose and disaccharides such as trehalose and melibiose. It was found to be EDTA sensitive (at 30 mM), pH-dependent (between 6–9) and heat-labile (upto 60 °C). A significant homology for soursop lectin was recorded to leguminous seed lectin from Psophocarpus scandens in MALDI-TOF-MS analysis with 66 % of sequence coverage. Finally, the toxicity bioassay of soursop lectin resulted in 100 % larval mortality for A. aegypti at 48 h whereas only 10 % mortality recorded to non-target organism C. costatus. Therefore, we concluded that soursop lectin could be a potent insecticidal agent in integrated pest management for controlling various insect pests.     

Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

A d-galactose-binding lectin purified from coronate moon turban,Turbo (Lunella) coreensis,with a unique amino acid sequence and the ability to recognize lacto-series glycosphingolipids

A divalent, cation-independent d-galactose-binding lectin was purified from coronate moon turban Turbo (Lunella) coreensis. This lectin recognizes d-galactose and is a 38-kDa dimeric protein consisting disulphide-bonded 22-kDa polypeptides under non-reducing and reducing conditions of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Haemagglutination activity was inhibited by d-galactose, N-acetyl d-galactosamine, melibiose, lactose, porcine stomach mucin, asialofetuin and bovine submaxillary mucin. The lectin has tolerance for pH 5–11 and temperature until 50 °C for 1 h. The lectin strongly aggregated Gram-negative bacteria, such as Vibrio parahaemolyticus and Salmonella O7, but weakly Gram-positive strain as Staphylococcus aureus and Bacillus subtilis. The glycan-binding profile of this lectin was evaluated using frontal affinity chromatography technology and the lectin appeared to recognize oligosaccharides such as lacto-series glycosphingolipids contained in blood type A and H substances in addition to complex-type N-linked glycoproteins. Partial primary structures of 139 amino acid residues of this lectin were determined from N-terminus polypeptides and 8 peptides derived by cleavage with lysyl-endopeptidase. The primary structure was slightly similar to other known sequences of lectin; however, a repeating motif has been included.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Datura stramonium Agglutinin : Location in the Seed and Release upon Imbibition

The distribution of Datura stramonium agglutinin over different tissues of D. stramonium L. seeds was visualized by immunocytochemical techniques and quantified by agglutination assays. The lectin occurs predominantly in the outer seed tissues (seed coat and seed epidermis), where it is associated, at least in part, with the cell walls. Developing D. stramonium seeds secrete newly synthesized lectin polypeptides into the incubation medium, which confirms the extracellular location of the lectin. Imbibition of mature decoated seeds results in a rapid and highly specific release of lectin. Indeed, imbibition solutions contain almost exclusively the lectin together with a few other carbohydrate-binding proteins; this is indicative of the predominance of these proteins in the seed surface layer. The presence of important amounts of lectin in the outer tissues of the seed is consistent with a possible role in the mediation of cell-cell interactions.     

Characterization of a lectin from the seeds of Erythrina vespertilio

A lectin was isolated from the seeds of Erythrina vespertilio by affinity chromatography on lactose-Sepharose 6B. The lectin has an M, of 59 000 and consists of two non-covalently associated subunits (M, ∼ 30 000). The lectin is devoid of cysteine but has six methionine residues/mol and a neutral sugar content of 9.7% The carbohydrate composition was mannose, N-acetylglucosamine, fucose, xylose and galactose in amounts of 15.0, 4.0, 1.0, 5.0 and 25 mol/59 000 g, respectively. Alkaline gel electrophoresis and isoelectric focusing showed that the affinity purified lectin consists of a family ofisolectins. Valine was the only N-terminal amino acid found and the N-terminal sequence was homologous with that found for other legume lectins. The lectin was inhibited by galactosyl containing carbohydrates; p-nitrophenyl-β-galactoside was the best inhibitor and the lectin showed a slight preference for β-galactosides. Comparison of its properties with those of other Erythrina lectins shows that most of the lectins of this genus are closely related.     

Lectin in Vegetative Tissues of Adult Barley Plants Grown under Field Conditions

A study of the distribution of lectins over different vegetative tissues of barley (Hordeum vulgare L.) plants, which were grown under normal crop conditions, indicated that lectin occurs in roots, leaves, and developing ears. Isolation and characterization of both root and leaf lectins led to the conclusions (a) that they are indistinguishable from the embryo lectin and (b) that the total lectin content of these vegetative organs is many times higher than that of the embryo. Finally, in vivo labeling experiments demonstrated that the lectin is synthesized de novo in roots and leaves.     

Multiple molecular forms of peanut lectin: Classification of isolectins and isolectin distribution among genotypes of the genus Arachis

Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V₂ types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of M_r 30,000 and a second polypeptide of M_r 60,000. Lectin preparations from five genotypes lack the M_r 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic.     

Purification and characterization of a galactose-specific lectin from corn (Zea mays) coleoptyle

We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl–Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since β-galactose residues are better recognized than those from the anomeric α-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.     

Lectins and the soybean-Rhizobium symbiosis: I. Immunological investigations of soybean lines,the seeds of which have been reported to lack the 120 000 dalton soybean lectin

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunofiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lactin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography.Phosphate buffered saline extracts of roots, hypocotyls, stems, and leaves of 3–66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 μg soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were modulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.     

Further characterization of the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, named carcinoscorpin, has been isolated from the horseshoe crab Carcinoscorpius rotunda cauda. It is a glycoprotein of molecular-weight 420,000, having two subunits of molecular weight 27,000 and 28,000, both subunits responding to glycoprotein stain. Leucine was detected as the only NH₂-terminal amino acid. The sedimentation constant of the native lectin was found to be 12.7 s. On digestion with trypsin, the lectin gave 18 soluble tryptic peptides. This lectin was found to be antigenically unrelated to another sialic acid-binding lectin, limulin, isolated from the horseshoe crab Limulus polyphemus. A lectin-specific disaccharide alcohol namely O-(N-acetylneuraminyl) (2 → 6)2-acetamido-2-deoxy-d-galactitol was found to quench the typical tryptophan fluorescence of the native lectin at 332 nm. The association constant for this interaction was determined spectrofluorimetrically and found to be 1.82 × 10³m^?1.     

Plant protoplast agglutination by lectins

Concanavalin A, soybean (Glycine max L.) lectin, castor bean (Ricinus communis L.) lectin, and peanut (Arachis hypogaea L.) lectin were able to agglutinate protoplasts prepared from a variety of plant species. The seven other lectins tried were unable to agglutinate those protoplasts tested. Protoplasts prepared from 11 species were used. The lectins examined were not able to differentiate among protoplasts of different species.     

Isolation, purification, and partial characterization of a lectin from chinook salmon ova

Chinook salmon (Oncorhynchus tshawytscha) ova contain a lectin that agglutinated human type B and rabbit erythrocytes and was specifically inhibited by the monosaccharides d-galactose and l-rhamnose. The lectin purified from homogenates of the ova by affinity chromatography on agarose possessed a pI of 4.5 in isoelectric focusing studies. The purified lectin inhibited the growth of four bacterial fish pathogens.     

Photosynthetic rate and lectin activity of soybean leaves after inoculation with rhizobia together with homologous lectin

Nodule bacteria (Bradyrhizobium japonicum) of various activities were preincubated with homologous lectin and then used for inoculating soybean (Glycine max (L.) Merrill) seeds. The effect of this inoculation on the photosynthetic rate, lectin activity in leaves, and plant development at different supply of mineral nitrogen was investigated under the conditions of pot experiments. There was a positive relationship between the photosynthetic rate and the lectin activity of proteins isolated from soybean leaves. Under the conditions of effective symbiosis, activation of functioning of the symbiotic apparatus by preincubation of the rhizobia with lectin exerted an additional stimulating effect on the photosynthetic rate. It is suggested that a relationship between the effectiveness of legume-rhizobium symbiosis and the lectin activity in leaves is mediated by the regulation of photosynthesis through a demand for assimilates in the source-sink system of soybean plants.