Immunoblotting detection of lectins in gluten and white rice flour

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour.     

A new type of cereal lectin from leaves of couch grass (Agropyrum repens)

Extracts from couch grass (Agropyrum repens) leaves contain relatively high lectin concentrations. Preliminary experiments with crude extracts indicated that the leaf lectin differs from the embryo lectin of the same species and other Gramineae embryo lectins with respect to its sugar and blood group specificity, and serological properties. A comparison of the biochemical, physicochemical and biological properties of purified lectins from couch grass leaves and embryos, and wheat germ agglutinin revealed that the leaf lectin has the same molecular structure as the embryo lectins. It is a dimer composed of two identical subunits, which, however, are slightly larger than embryo lectin subunits. Structural differences between both couch grass lectins were further inferred from in vitro subunit exchange experiments and serological analyses. Whereas the embryo lectin readily forms heterodimers with embryo lectins from other cereal species and also is serologically indistinguishable from them, the leaf lectin does not exchange subunits with the same embryo lectins and is serologically different. In addition, couch grass leaf lectin exhibits specificity for N-acetylgalactosamine and agglutinates preferentially blood-group-A erythrocytes whereas the embryo lectin is not inhibited by N-acetylgalactosamine and exhibits no blood-group specificity. It was observed also that the lectin content of couch grass leaves varies enormously during the seasons.     

Immobilization of immunoglobulins using lectins with an immune sensor method based on surface plasmon resonance

The effect of different lectins upon the response of immune sensor based on surface plasmon resonance (SPR) was investigated. Lectins affinity to carbohydrates of the IgG can be used to increase the density and orientation of IgG molecules at their immobilisation on the sensor surface. Conditions were elaborated for enhancement of immune sensor response in comparison with that one for bare or preliminary treated with dodecanthiol thin gold sensor surface. It was shown that human IgG revealed maximal affinity to wheat germ lectin and mouse monoclonal antibodies and rabbit IgG--to helix pomatia lectin. Pig antibodies, similar to human IgG, showed the greatest affinity to wheat germ lectin.     

Biosynthesis of lectin in roots of germinating and adult cereal plants

Root tips of wheat, rye, barley and rice seedlings contain lectins which are identical to the respective embryo lectins with respect to their molecular weight, sugar-specificity and serological properties. Using in vivo labelling techniques, it could be demonstrated that lectin is synthesized de novo in these tissues. The presence of lectin mRNA in seedlings was confirmed by in-vitro synthesis of lectin in root-tip extracts. Lectin synthesis occurs both in primary and first adventitious roots and is confined to the apical part (2mm) of the root. As seedling development proceeds, lectin synthesis in root tips gradually decreases. Adventitious roots of adult (five to six months old) wheat, rye and barley, but not rice, plants also contain lectins which are indistinguisable from the embryo lectins by the above-mentioned criteria. These lectins are synthesized in vivo in isolated root tips (5 mm) with labelled cysteine and in vitro in cell-free extracts prepared from root tips. Synthesis of lectin in roots of adult plants is also confined to the apical (2 mm) tip of the roots. At the molecular level, root lectin synthesis is very similar to that in embryos. All root lectins are synthesized as 23 000-M_r precursors which are post-translationally converted into the mature 18 000-M_r polypeptides. The observation that seedling roots and adventitious roots of six-month-old plants actively synthesize lectins strongly indicates that lectin genes are expressed in these tissues. In addition, since the root lectins are indistinguishable from the embryo lectins, we postulate that the same lectin genes are expressed.Abbreviations ABA abscisic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

Immunological cross-reactivity between beta-galactoside-binding lectins of vertebrates. Possible relationship of antigenicity to oligomeric structure

Structural relationships among five beta-galactoside-binding lectins isolated from human, mouse and chick were studied using immunochemical methods. The lectins examined were human placenta lectin with a 14-kDa subunit (human 14K lectin), two types of mouse lectin (mouse 15K and mouse 16K lectin), and two types of chick lectin (chick 14K and chick 16K lectin). Five polyclonal antibodies raised against these lectins were used. Antibody to human 14K lectin cross-reacted with mouse 15K and chick 14K lectins. Antibodies to both mouse 15K and chick 14K lectins cross-reacted with human 14K and chick 16K lectins. Antibody to chick 16K lectin cross-reacted with mouse 15K lectin. An immunological relationship was not found between human 14K and chick 16K lectins, or between mouse 15K and chick 14K lectins. Mouse 16K lectin did not show any immunological relationship with any of the other lectins. A monoclonal antibody raised against chick 14K lectin cross-reacted with chick 16K lectin. These results cannot be explained simply in terms of phylogenic distance but suggest that vertebrate beta-galactoside-binding lectins can be classified into two structural groups on the basis of their antigenicities. One group, which is characterized as a monomer type, includes human 14K and chick 14K lectins. The other group, which is characterized as a dimer type, includes mouse 15K and chick 16K lectins.     

Effects of lectins with different carbohydrate-binding specificities on hepatoma, choriocarcinoma, melanoma and osteosarcoma cell lines

The effects of lectins with different carbohydrate-binding specificities on human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma (B16) and rat osteosarcoma (ROS) cell lines were investigated. Cell viability was estimated by uptake of crystal violet. Wheat germ lectin was the lectin with the most deleterious effect on the viability of H3B, JAr and ROS cell lines. The cytotoxicity of lectins with similar sugar-binding specificity to wheat germ lectin, including Maackia amurensis lectin and Solanum tuberosum lectin, was weaker than that of wheat germ lectin. N-acetylgalactosamine-and galactose-binding Tricholoma mongolicum lectin ranked third, after wheat germ lectin and Maackia amurensis lectin, with regard to its effect on H3B, and ranked, together with Maackia amurensis lectin, as the lectins with the second most pronounced effects on ROS. However, the cytotoxic effects of Tricholoma mongolicum lectin on JAr were much weaker than those of Maackia amurensis lectin, Solanum tuberosum lectin and Anguilla anguilla lectin. Artocarpus integrifolia lectin, Lens culinaris lectin and Anguilla anguilla lectin possessed milder cytotoxicity than the remaining lectins. which were approximately equipotent. The mannose-binding Narcissus pseudonarcissus and Lens culinaris lectins were only weakly cytotoxic, the exception being a stronger effect on H3B. The N-acetylgalactosamine-binding Glycine max lectin and methylgalactose-binding Artocarpus integrifolia lectin similarly exhibited low cytotoxicity. It can thus be concluded that in general the ranking was wheat germ lectin > Maackia amurensis lectin approximately Trichloma mongolicum lectins > other aforementioned lectins in cytotoxicity. A particular lectin may manifest more conspicuous toxicity on certain cell lines and less on others.     

植物凝集素在植物体内的生理作用

现有研究表明,植物种子中的凝集素是植物体内的储存蛋白;扁豆和稻胚凝集素对胚胎的分裂和分化有促进作用;在豆科植物和根瘤菌之间的共生作用中,凝集素起着高度专一的识别作用;麦胚凝集素在种胚萌发时,起着抗真菌的作用;体外实验也证明凝集素对危害玉米的主要害虫的发育有阻碍作用;还发现植物凝集素具有酶的活性和酶抑制剂的作用,从而调节植物体的生理活动。     

Occurrence and immunological relationships of lectins in gramineous species

A survey of the occurrence of lectins in seeds from more than 100 grass species showed that all species belonging to the Triticeae tribe and the genera Brachypodium and Oryza contain lectins. All these lectins have the same sugar-binding specificity and are related to wheat-germ agglutinin, but to different degrees. Lectins from Triticeae species are immunologically indistinguishable from wheat lectin, whereas Brachypodium and rice lectins are only immunologically related to the wheat lectin. Attempts to detect lectin-deficient lines or varieties in wild and cultivated species of the three lectin-containing groups were unsuccessful. The possible use of lectins as a chemotaxonomic tool is discussed.     

Interrelationships between Gramineae lectins

Two Gramineae species, oat and maize, are compared with wheat and barley to see if they contain lectins which are structurally and functionally similar to the Hordeae lectins. Four distinct criteria were examined: localisation of lectin activity in the seed, ability to agglutinate a defined type of erythrocyte in a reaction reversed by monomers or oligomers of n-acetyl-d-glucosamine, ability to bind to the affinity matrix p-aminobenzyl-1-thio-2-acetamido-2-deoxy-β-d-glucopyranoside-substituted Sepharose, and cross-reactivity with monospecific antisera raised to wheat-germ agglutinin. Results indicate that the very close relationship found between the lectins of wheat, barley and rye cannot be extended to those species of Gramineae outside the tribe Hordeae.     

Occurrence and synthesis of lectin in coleoptiles of germinating wheat,rye and rice seedlings

Occurrence, synthesis and localization of lectins in coleoptiles of 3-day old seedlings of wheat, rye, barley and rice were studied by a combination of high resolution ion-exchange chromatography, in vivo labelling with ³⁵S-cysteine and immunocytochemistry. Whereas no lectin can be isolated or localized in barley coleoptile, 1.9 and 40 ng of lectin per coleoptile was obtained from wheat and rye respectively. Wheat germ agglutinin was localized in the outer layer of the wheat coleoptile and both inner and outer layers of rye coleoptile displayed a specific reaction. In rice, 250 ng of lectin is present in the coleoptile and is distributed throughout this organ. Wheat coleoptiles synthesize no lectin and rye coleoptiles synthesize minute amounts while those from developing rice seedlings incorporate reasonable amounts of ³⁵S-cysteine into lectin.Abbreviations FPLC Fast Protein Liquid Chromatography - GlcNAc N-acetylglucosamine - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Isolation, molecular and biological properties of a lectin from rice embryo: relationship with wheat germ agglutinin properties

Rice lectin (Oryza sativa, var. Balilla 28) was purified from defatted embryos by aqueous acid extraction at pH 1.3 followed by ammonium sulfate precipitation between 2 and 4 M, affinity chromatography on agarose-p-aminophenyl-beta-D-N-acetylglucosamine, and gel filtration on AcA 54. The homogeneity of the lectin was checked by polyacrylamide gel electrophoresis, gel filtration, and immunodiffusion. The amino acid analysis revealed a high half-cystine content (9%) and a low aromatic and hydrophobic amino acid content. The lectin contained neither neutral carbohydrates nor amino sugars. The isoelectric point was estimated to be 8.1. The molecular weight of rice lectin was estimated to be 38,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions showed two polypeptides with Mr 19,000 and 15,000. The circular dichroism spectrum of rice lectin in far ultraviolet was characterized by a positive maximum at 228 nm and a negative band at 203 nm suggesting the presence of a beta-pleated sheet and the absence of alpha-helix. Rice lectin had no human blood group specificity and agglutinated rabbit erythrocytes more efficiently than erythrocytes from other animal species. Furthermore, agglutination was enhanced by trypsin treatment of erythrocytes. The erythroagglutinating activity was very high since the minimal concentration needed to agglutinate erythrocytes was 0.05 micrograms/ml. Although [methyl-3H]thymidine incorporation was stimulated in human lymphocytes, rice lectin could not be considered as a mitogenic lectin since it stimulated neither blast transformation nor lymphocyte proliferation. The saccharide specificity of rice lectin was related to N-acetylglucosamine and its oligomers: N,N',N"-triacetylchitotriose was the most powerful inhibitor. Furthermore, the N-acetylneuraminic acid was not a specific rice determinant. Finally, the double immunodiffusion method revealed a cross-reactivity between rice lectin and wheat germ agglutinin, indicating that these lectins were closely antigenically related. The analogies and differences between biological and immunological properties of rice lectin and wheat germ agglutinin are discussed and the possibility of their evolution from a common ancestor is put forward.     

Studies on lectins. LVII. Immunofluorescence localization of lectins present in fish ovaries

The occurrence of endogeneous lectins in the ovaries of four fish species has been studied by indirect immunofluorescence staining with antibodies against individual lectins. Paraffin sections of the ovary of perch (Perca fluviatilis L.) were treated with an antibody against perch lectin. In cryostat sections of the tench (Tinca tinca L.) ovary, the L-rhamnose-specific lectin "I" was detected with a specific antibody. In cryostat sections of both roach (Rutilus rutilus L.) and rudd (Scardinius erythrophthalmus L.) ovaries, lectins were localized using a single antibody against roach lectin. The isolation of tench lectins is briefly described. In the fish species employed for this study, lectins are associated exclusively with the content and surrounding membrane of cortical vesicles situated within the cytoplasm of maturing oocytes. The positive reaction with lectin antibody was observed almost immediately after the formation of the first cortical vesicles in the peripheral cytoplasm of early previtellogenic oocytes. Their lectin content increases during the later stages when cortical granules fill the whole cytoplasm before moving towards the cell periphery, as the oocyte starts to accumulate yolk. The presence of lectins within cortical vesicles is significant also in view of the polysaccharide content of these structures. In the vitellogenic oocytes lectins seem to move towards the cell periphery and accumulate beneath the plasma membrane. Our observations are discussed in view of the present ideas on the intracellular function of lectins, and with respect to the role of cortical vesicles in fertilization and in post-fertilization modifications of the egg envelopes.     

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.     

Localization of soluble endogenous lectins and their ligands at specific extracellular sites

Soluble lectins of chicken, rat, frog, and the cellular slime mold, Dictyostelium discoideum, were purified and specific antibodies raised against these proteins were used to immunohistochemically localize the lectins in and around the tissues in which they were synthesized. Within cells, some of these soluble lectins (chicken-lactose-lectin-II in intestinal goblet cells, discoidin II in prespore cells) appear to be concentrated within vesicles whereas others (e.g., rat beta-galactoside lectin in pulmonary alveolar and smooth muscle cells) appear to be free in the cytoplasm. All of these lectins are eventually secreted to extracellular sites in developing or adult tissues. The sites include mucin (chicken-lactose-lectin-II in intestine); developing extracellular matrix (chicken-lactose-lectin-I in muscle; Xenopus laevis lectin in blastula stage embryos); slime (discoidin I); developing spore coat (discoidin II); and a specialized extracellular matrix, elastic fibers (rat beta-galactoside lectin in lung). In cases where this has been studied in detail (discoidin I, discoidin II, and chicken-lactose-lectin-II), the lectin is associated with a complementary extracellular ligand, at least transiently. Lectin-ligand interactions presumably confer specialized properties in these particular extracellular domains.     

Reactivity with legume lectins of three monoclonal antibodies made against the Lathyrus odoratus lectin

Three murine IgG1 monoclonal antibodies (MoAb) were produced against the glucose/mannose-specific two-chain mitogen from seeds of Lathyrus odoratus (sweet pea) belonging to the Vicieae tribe of the Leguminosae family. Their antigenic specificities were tested against subunits of two-chain and one-chain legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. Different binding to native and detergent-treated lectins in dot-blots indicated that two of the antibodies bound to conformational epitopes which by immunoblotting were shown to be expressed on the heavy subunits from all the five tested two-chain lectins and on the six one-chain lectins, including PHA (Phaseolus vulgaris) and Con A (Canavalia ensiformis). However, ELISA cross-inhibition studies showed that these two antibodies bound to different epitopes on the lectin molecules. The third MoAb reacted with a continuous epitope not revealed by classical taxonomy since only three of five heavy subunits of the two-chain lectins stained with the antibody. Comparison of the known amino-acid sequences of the chains indicates eight positions as possible binding sites for this antibody.     

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     

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests,Nilaparvata lugens andNephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.     

Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.