Bean Lectin Research Articles

Specificity of adenine binding to lima bean lectin

The interactions between lima bean lectin (LBL) and adenine were examined using a series of synthetic purine analogs. Binding was sensitive to modification at most positions of the purine ring, suggesting a high degree of specificity for adenine binding. Methylation ofthe 6 NH 2-group to MeNH-, Me 2N- and Me 3N +-analogs progressively decreased the binding affinity. Compounds lacking the 6 NH 2-group were not bound. Methylation of adenine at N 1, N 3 or N 7 also inhibited binding, indicating specific interactions with these ring nitrogens. In contrast to the previous report that N 9-substituted adenines, nucleosides and nucleotides were not bound [Roberts, D. D. and Goldstein, I. J. (1983) J. Biol. Chem. 258, 13820], 9-methyl- and 9-benzyl-substituted adenines were bound to LBL with high affinity. Substitutions at C-2 and C-8 were tolerated and, in some cases, increased the affinity of binding to LBL. Heterotropic interactions between the adenine and 1,8-anilinonaphthalenesulphonate binding sites were also sensitive to modification of the purine ring. 2-Methylthioadenine and 4-aminopyrazolo[3,4- d]pyrimidine showed increased allosteric interaction with 1,8-anilinonaphthalenesulphonate binding, whereas several adenine analogs with a 9- p-nitrobenzyl substituent appeared to be negative effectors of 1,8-anilinonaphthalenesulphonate binding.

Effect of Ingested Toxic Bean Lectins on the Gastrointestinal Tract in the Rat

The present study was undertaken to provide further evidence for the mechanisms proposed for the toxicity of ingested bean lectins in animals: 1) to show the stability of concanavalin A (Con A) in the gastrointestinal tract so that it has enough time to interact with some enzymes localized in the intestinal membrane and 2) to find its effect on the activities of those enzymes that have been adopted as criteria for adaptive changes in response to altered diets, namely intestinal sucrase, alkaline phosphatase and leucine aminopeptidase. Significant amounts of ingested Con A were recovered unaltered (as seen from affinity chromatography and electrophoresis) from the cecal content of rats 4 h after its oral administration and from feces (90% recovery) 4 d later. This indicated that Con A is quite stable during its passage through the gastrointestinal tract. Con A, given at a level of 0.3 or 0.5% in the diet, completely prevented adaptive changes in the activities of those enzymes. These results substantiate the mechanisms proposed earlier by other investigators that the toxicity of ingested bean lectins involves their binding to the luminal surface of the small intestine, where they disturb the function of the brush border membrane.

In vitro endoproteolytic cleavage of castor bean lectin presursors

The toxic (A) and cell binding (B) chains of the castor bean lectins ricin and Ricinus communis agglutinin are synthesized together in the form of single polypeptide precursors. The precursors are transported in vivo to the protein bodies where an endoproteolytic step generates the individual lectin subunits. We have demonstrated an acid endopeptidase activity from developing castor bean endosperm or from protein bodies isolated from mature seeds which cleaves the precursors to release the subunits in vitro.

Molecular model for the complex between concanavalin A and a biantennary‐complex class glycopeptide

A molecular model for the complex formed between the jack bean lectin concanavalin A (Con A) and glycopeptides of the complex biantennary class is described. The model was derived using coordinates for Con A determined by x-ray crystalographic refinement techniques, with 1.75-A resolution data, and coordinates for the glycopeptides obtained from 1H-nmr measurements, using the nuclear Overhauser effect. Previous solution and crystallographic studies provided several constraints on the possible mode of interaction of the lectin and the glycopeptide. Examination of the model suggests that the glycopeptide binding site is defined by four loops on the protein surface made up by amino acid residues: 12–18, 98–102, 205–208, and 226–229. Within these loops, it favorable interactions with high-affinity ligands and tose responsible for the unfavourable interactions with poor ligands.

Lectins in foods and their relation to starch digestibility

Lectin concentrations of 16 foods and total lectin intakes in test meals were related to glycemic responses to the foods in normal and diabetic individuals. Lectin concentration showed a significant negative relationship with glycemic response in both normal and diabetic individuals suggesting that lectins may play a role in determining the rate of digestion of carbohydrate. Similar relationships were also seen between lectin content and in vitro rate of digestion indicating that lectins may affect the luminal stage of digestion. Kidney beans cooked for 30 min which contained lectins were digested more slowly in vivo than beans cooked for 60 min which contained no lectins. Both cooked beans had digestion rates lower than lectin free white bread. Kidney bean lectin added to white bread reduced the rate of starch digestion in vitro. Lectins may account for up to half the difference in digestibility between kidney beans and bread.

Binding of lectins to Streptococcus mitis cells. Studies of the specificity of ligand mediated aggregation.

Previous studies have shown that the mechanism of spontaneous aggregation of Streptococcus mitis ATCC 903 depends on a lectin-ligand type interaction. To study the specificity of the ligand, the binding of a number of lectins of different sugar specificities to the surface of untreated, trypsin and beta-galactosidase-treated bacteria was studied by assessing aggregation. Untreated bacteria were rapidly aggregated by concanavalin A (Con A), wheat-germ agglutination (WGA) and helix pomatia lectin (HPL). Other lectins tested, e.g. peanut agglutinin and soy bean lectin, did not induce aggregation. Lectin-induced aggregation was distinguished from the spontaneous one by recording the course of aggregation and inhibition of lectins by specific sugars. Trypsin-treated bacteria lost their ability for both spontaneous and lectin-induced aggregation. beta-galactosidase-treated bacteria were aggregated only in the presence of Con A and HPL. The bacteria retained their ability for spontaneous aggregation after removal of lectins and inhibitory sugars. These findings suggest that ligand is of glycoprotein nature, since it was removed from the bacterial surface by treatment with trypsin, as shown by the inability of treated cells for both spontaneous and lectin-induced aggregation. Partial degradation of the carbohydrate part of the ligand is indicated by the ability of beta-galactosidase-treated bacteria to aggregate in the presence of Con A and HPL.

Abnormal Processing of the Modified Oligosaccharide Side Chains of Phytohemagglutinin in the Presence of Swainsonine and Deoxynojirimycin

Phytohemagglutinin, the glycoprotein lectin of the common bean, Phaseolus vulgaris, has both high-mannose (Man(8-9)GlcNAc(2)) and modified oligosaccharide side chains. The modified side chains have glucosamine, mannose, fucose, and xylose in the molar ratios 2:3.8:0.6:0.5, and are resistant to hydrolysis by endoglycosidase H. Synthesis and processing of side chains in the presence of 1-deoxynojirimycin, an inhibitor of alpha-glucosidase, results in the formation of chains which are all alike. They are sensitive to endoglycosidase H, do not contain fucose, and are largely resistant to alpha-mannosidase. This indicates that they are probably high-mannose chains blocked by terminal glucose residues. Synthesis and processing of side chains in the presence of swainsonine, an inhibitor of alpha-mannosidase II, results in the formation of normal high-mannose chains, and of modified chains which contain fucose residues, are resistant to endoglycosidase H, and can be distinguished from normal modified chains only by the presence of extra mannose residues.Processing of the phytohemagglutinin modified chains of PHA under normal conditions involves the attachment of peripheral N-acetylglucosamine residues in the Golgi complex and their subsequent removal in the protein bodies. The attachment of the N-acetylglucosamine residues is largely inhibited by deoxynojirimycin but still occurs in the presence of swainsonine. The results presented in this work show that processing of the asparagine-linked oligosaccharides is under the control of several glycosidases and glycosyltransferases and involves the formation of intermediate products.

Nutritional response of mature rats to kidney bean (Phaseolus vulgaris) lectins

AbstractIn a series of 10‐day pair feeding experiments it was found that the nutritional value of diets containing beans was essentially the same for rats aged between 30 and 123 days. Thus net protein utilisation (NPU) values of 25–39 on diets containing Processor bean (35 g protein kg−1) + egg albumin (65g protein kg−1) were obtained. As food intakes were considerably reduced when rats were fed diets containing more than 35g protein kg−1 of Processor bean, the measurement of protein utilisation became increasingly more difficult. The severe disruption of the brush borders of duodenal and jejunal enterocytes, originally observed when bean‐containing diets were fed to young (30‐day‐old) rats was also found with rats up to the age of 120 days on similar diets. Similarly, the development of circulating anti‐lectin antibodies in the animals showed no age dependence within the age limits investigated. It was also shown that oral immunisation did not protect the rats from the effects of toxicity and that the immune response was a result of continuous absorption of lectin throughout the feeding period. Thus the extent and the mechanism of toxicity of Phaseolus vulgaris bean lectins were found not to be dependent on the age or maturity of the animal.

Bean lectins

Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F2 plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.

Nucleotide sequence of cloned cDNA coding for preproricin.

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).

Fractionation of Kintoki bean lectin into isolectins.

A comprehensive study of the preparative procedure of Kintoki bean lectin resulted in the resolution of the lectin into four isolectins whose pIs varied from 5.19 to 5.67. They agglutinated human, goat, hen and mouse erythrocytes, but not those of cow. The more acidic the isolectins, the less active were the erythrocyte agglutination and the more active the stimulation of sheep lymphocytes. Although the general patterns of amino acid composition were similar, characterized by higher contents of aspartic acid, leucine and valine and lack of sulfur-containing amino acids, differences were found in a few amino acids such as phenylalanine, valine and lysine. Each lectin seems to be a tetramer of a 33,000 dalton subunit which is thought to differ in charge from lectin to lectin.

Studies on the Agglutinability of Trypsin-treated Chicken Red Blood Cells with Soy Bean Lectin

Studies on the Agglutinability of Trypsin-treated Chicken Red Blood Cells with Soy Bean Lectin

Amphiphilic cationic drugs and phospholipids influence the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fraction of untreated rats.

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.

Protein blotting: Detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes

Methods to detect “native” proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield and indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.

Molecular model for the complex between Concanavalin A and a biantennary‐complex class glycopeptide

AbstractA molecular model for the complex formed between the jack bean lectin concanavalin A (Con A) and glycopeptides of the complex biantennary class is described. The model was derived using coordinates for Con A determined by x‐ray crystalographic refinement techniques, with 1.75‐Å resolution data, and coordinates for the glycopeptides obtained from 1H‐nmr measurements, using the nuclear Overhauser effect. Previous solution and crystallographic studies provided several constraints on the possible mode of interaction of the lectin and the glycopeptide. Examination of the model suggests that the glycopeptide binding site is defined by four loops on the protein surface made up by amino acid residues: 12–18, 98–102, 205–208, and 226–229. Within these loops, it favorable interactions with high‐affinity ligands and tose responsible for the unfavourable interactions with poor ligands.

Development of two assay systems of desialylated hCG specific for O-serine or N-asparagine linked carbohydrate chain and their clinical application

Two specific and sensitive assays have been developed for the measurement of as-hCG in this study. These assay systems are immunoradiometric assays which utilize peanut lectin, Arachis hypogaea (PNA) or caster bean lectin, Ricin communis agglutinin (RCA-I), to selectively extract as-hCG and then directly quantify with purified and radiolabeled antiserum against hCG beta COOH terminal peptide (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-GalNAc which is only exposed in hCG when the O-serine linked oligosaccharide of its unique beta-CTP region are desialylated. RCA-I is also specific for the terminal carbohydrate beta-D-Galp., especially the linkage of Gal beta-(1----4)-GlcNAc, which is exposed in hCG when the N-asparagine linked oligosaccharide of its alpha and beta subunits are desialylated. The assays (PNA-R525 and RCA-I-R525) are capable of reliably and accurately measuring as little as 0.01 and 0.002 pmoles as-hCG/ml without interference from hCG, respectively (crossreactivity: 0.1% and 0.03%). The urine concentrates of a patient with choriocarcinoma and a normal pregnant woman were fractionated by Sephadex G-100 gel filtration and total hCG and as-hCG levels were measured. When the fractions were analysed by beta-CTP RIA, there were two peaks of immunoreactive hCG in the urine of a patient with choriocarcinoma. The bigger molecular fractions were hCG of native size, and 16% of which was desialylated. The smaller molecular fraction was beta-CTP, and practically all of that was desialylated. Whereas, beta-CTP could not be observed, and only 1.5% of hCG of native size was desialylated in the urine of a normal pregnant woman. Although the absolute levels of as-hCG in the urine specimens increased as total hCG increased, the proportion of as-hCG decreased as total hCG concentration increased. The proportion of as-hCG in urine specimens from choriocarcinoma patients was higher than specimens from patients with hydatidiform mole (including invasive mole) in both these lectin immunoradiometric assay (LIRMA) systems. Also, the proportion of as-hCG in urine specimens from molar patients was higher than those from normal pregnant women using PNA as extracting reagent. Whereas, when RCA-I was used, there was no significant difference between specimens from molar patients and those from normal pregnant women.(ABSTRACT TRUNCATED AT 400 WORDS)

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport.

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.

Binding between pole bean (Phaseolus vulgaris L.) lectin and rhizobia that do or do not nodulate the pole bean

The pole bean (Phaseolus vulgaris L. cv. Kentucky Wonder) was nodulated not only by Rhizobium phaseoli 127K14 but also by Rhizobium sp. 127E15, R. leguminosarum 92A3, and R. trifolii 0403. It was not, however, nodulated by R. trifolii ATCC 14481 and R. japonicum 61A118. The binding between pole bean lectin (PBL) and various strains of Rhizobium that can or cannot nodulate the pole bean was tested. Using rabbit antibodies prepared against purified PBL, the lectin was shown by immunofluorescence microscopy to be present on the pole bean root hair surface. The binding between PBL and exponentially growing rhizobia was quantified by a radial immunodiffusion technique. PBL was found to bind to all rhizobia tested whether they nodulate the pole bean or not. The amount of PBL bound to R. phaseoli 127K14, Rhizobium sp. 127E15,R. leguminosarum 92A3, R. trifolii 0403, R. trifolii ATCC 14481, and R. japonicum 61A118 was 3.7, 4.0, 7.4, 5.7, 7.8, and 6.5 μg per 109 cells, respectively. Although hemagglutination activity of PBL was inhibited by N-acetyl-D-galactosamine, the binding betwen PBL and rhizobia was strongly inhibited by D-gluconate, D-galacturonate, and polygalacturonate. The latter three carbohydrates did not inhibit hemagglutination. The results suggest that the sugar specificity of PBL for binding rhizobia may be different than that for the interaction with erythrocytes. Since the sugar specificities of PBL and soybean lectin for binding R. japonicum are different, rhizobia such as R. japonicum 61A118 may have multiple independent binding sites, each having different sugar specificity, for lectins of various legumes.

Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds

Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined.