Purification and characterization of mannose/glucose-specific lectin from seeds of <Emphasis Type="Italic">Trigonella foenumgraecum</Emphasis>

A lectin present in seeds of Trigonella foenumgraecum was isolated and purified by acid precipitation, salt fractionation, and affinity chromatography on mannan cross-linked agarose. SDS-PAGE revealed a single band corresponding to a molecular weight of 27,350 daltons. The lectin agglutinated trypsin-treated rat erythrocytes. Sugar specificity as determined by hemagglutination inhibition assay indicated that the lectin belongs to a glucose/mannose-specific group. The reaction of the lectin with glycoprotein was affected by pH changes. The carbohydrate binding specificity of the lectin was investigated by turbidity and activity measurements. As the lectin belongs to the Leguminoceae family, the specificity of the lectin for glucose/mannose renders it a valuable tool for Rhizobium-legume symbiosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 52–57.     

A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.     

Histochemical identification and behavior of quail primordial germ cells injected into chick embryos by the intravascular route.

The behavior of quail primordial germ cells (PGC) after injection into chick embryos by the intravascular route was examined. The quail (donor) PGC, taken from the bloodstream of quail embryos (recipient) at stage 13-14, were injected into the vitelline vessels of chick embryos (recipient) at stage 15. In the recipient embryos, the PGC of the quail and the chick were histochemically distinguished by a double-staining technique involving a lectin, from Wistaria floribunda (WFA) and the PAS reaction. One day after injection, quail PGC appeared in the prospective gonadal region of recipient chick embryos, being localized among the recipient chick PGC. This result indicates that a staining technique specific for WFA lectin is useful for identification of quail PGC and that quail PGC can be transferred by a vascular route for the production of germline chimeras.     

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

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

7-Deoxyloganin 7-hydroxylase in Lonicera japonica cell cultures

A lectin was isolated from the mushroom Mycoleptodonoides aitchisonii by means of affinity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel filtration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of four identical 16 kDa subunits and the molecular mass of the intact lectin was estimated to be 64 kDa by gel filtration. In a hemagglutination inhibition assay, it exhibited strong sugar-binding specificity towards asialo-BSM among the mono- or oligo-saccharides and glycoproteins tested. The binding specificity of the lectin was also examined by surface plasmon resonance analysis.     

青山羊（Capra，hircus）小肠凝集素的分离、纯化及性质研究

报道了青山羊小肠凝集素的分离、纯化及性质研究。青山羊小肠先经过含有巯基乙醇的磷酸缓冲液抽提,然后上Ｓｅｐｈａｒｏｓｅ６Ｂ柱及ＤＥＡＥ－Ｃｅｌｌｕｌｏｓｅ－２３柱,得到纯化的青山羊小肠凝集素。采用ＳＤＳ电泳法测得其分子量在６６１００左右,而且该凝集素不含糖,对人Ｂ型血球有专一性凝集作用。半抗原抑制实验表明它对半乳糖（乳糖）有亲和性。其中酸性氨基酸含量较高,组氨酸、蛋氨酸含量较低。该凝集素在胚胎期出现,出生后几个月达到高峰然后逐渐下降,最后消失。     

Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.     

Purification and characterization of a magnesium ion requiring N-acetyl-D-glucosamine specific lectin from seeds of Quercus ilex L

A new magnesium ion requiring N-acetyl-D-glucosamine specific lectin QIL was purified to electrophoretic homogeneity from seeds of Quercus ilex L. through successive steps of (i) lectin extraction, (ii) ammonium sulphate (30-50%) fractionation, (iii) diethylaminoethyl (DEAE)-cellulose chromatography, (iv) carboxymethyl (CM)-cellulose chromatography, and (v) Sephadex G-75 chromatography. The lectin, having specific activity of 25,600 hemagglutination units (HAU)/mg of protein, was found to be a monomeric protein with a native molecular weight of 13.2 kDa. N-Acetyl-D-glucosamine was found to exhibit most potent inhibitory action on the lectin activity among all the sugars tested. The lectin was also found to exhibit specificity for human blood groups A, B, and AB. It was converted to the corresponding apo-lectin by ethylenediaminetetraacetic acid (EDTA) treatment followed by buffer dialysis. The apo-lectin exhibited a specific and characteristic requirement for magnesium ions for the expression of its activity.     

Chicken lactose lectin: Cell-to-cell or cell-to-matrix adhesion molecule?

Summary Endogenous chicken muscle lectin isolated by lactose affinity chromatography inhibits myoblast fusion. Similar lectins isolated from embryonic brain, heart, and liver and from adult intestine exhibit the same ability. Elevated levels of any of these lectins canceled the inhibitory effect. Peanut agglutinin isolated by the same procedure had no effect at any concentration tested. Concanavalin A affected fusion only at high concentrations. Muscle lectin was shown to agglutinate myoblasts in microtiter plates, whereas exogenous addition in culture inhibited alignment as seen by time lapse microcinematography. Cell-to-cell communication between lectin-treated cells was shown by nucleotide exchange, and lectin-coated culture dishes did not affect cell attachment. Our evidence shows a lack of specificity to muscle, but suggests an aggregating capacity between cells, or possibly an interaction between the cell membrane and the extracellular matrix. This study was supported by National Institutes of Health grants AM 25202 and HD 07104, The Muscular Dystrophy Association, and the Cleveland chapter of Sigma Xi.     

Isolation and characterization of a soybean lectin having 4-O-methylglucuronic acid specificity

A new lectin from soybeans having specificity toward the extracellular 4-O-methyl-D-glucurono-L-rhamnans produced by certain strains of Rhizobium japonicum has been purified and characterized. Isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose. Residual globulins were removed by adsorption of the lectin on concanavalin A-agarose and elution with methyl alpha-mannoside. The lectin is a glycoprotein (3-5% carbohydrate) with a molecular weight of approximately 175 000. It is a tetramer with subunit molecular weights of 45 000 when dissociated with sodium dodecyl sulfate. Reverse-phase high-pressure liquid chromatography analysis indicates the presence of two types of subunits, both having equivalent molecular weights. According to amino acid analyses, the lectin is rich in acidic but low in sulfur-containing amino acids. The carbohydrate portion of the lectin contains mannose; no hexosamines could be detected. Chemical modification of the lectin indicated that neither sulfhydryl groups nor amino groups participate in binding. Quantitative binding studies of the lectin with various carbohydrate haptens showed that specificity was directed toward 4-O-methyl-D-glucuronic acid, D-glucuronic acid, and their methyl glycosides with 4-O-methyl-D-glucuronic acid 3-4-fold more effective. In each instance, the methyl glycoside is a more effective hapten.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

Purification of Cajanus cajan root lectin and its interaction with rhizobial lipopolysaccharide as studied by different spectroscopic techniques.

A lectin present in roots of Cajanus cajan seedlings was isolated and purified by affinity chromatography. Sugar specificity assayed by hemagglutination-inhibition activity indicated that lectin belongs to glucose/mannose-specific group. The root lectin was found to be mannose-specific from the second day onwards as it was reconfirmed by specific elution of different days' sample from mannose agarose matrix. The maximum interaction of lectin with goat IgM was obtained in 10-day-old sample, indicating the highest crude lectin content. Lectin (total amount of eluted protein) from different days soil sample showed a maximum amount in 10-day-old sample. For further studies, the lectin has been isolated from the roots of 10-day C. cajan seedlings and purified on mannose-CL agarose column by affinity chromatography. Lectin was found to be a dimer of 18.5-kDa subunit as revealed by SDS-PAGE. Tryptophan quenching fluorescence was studied for C. cajan root lectin. Secondary structure of C. cajan root lectin as studied by circular dichroism was found to be a typical beta-pleated sheet structure. The interaction of purified root lectin with C. cajan-specific rhizobial lipopolysaccharide and its inhibition by specific and nonspecific sugars was demonstrated by fluorescence and circular dichroism. Results discussed in this paper were studied for the first time by different spectroscopic methods, suggesting that C. cajan root lectin-lipopolysaccharide interaction is specific.     

Lectin fromCanavalia brasiliensis (MART.). isolation,characterization and behavior during germination

A lectin was isolated fromCanavalia brasiliensis Mart. seeds by combining solubility fractionation with affinity chromatography on Sephadex G-50. The lectin showed a carbohydrate specificity for D-mannose (D-glucose) binding and a requirement for Ca²⁺ and Mn²⁺. All the hemagglutinating activity was found in the cotyledons and the presence of the lectin was followed during the first 15 days of plant germination, through the activity against rabbit erythrocytes, the presence of the “lectin peak” in Sephadex G-50 affinity chromatography, presence of the “lectin bands” in SDS-polyacrylamide gel electrophoresis and the “lectin arcs and rockets” in immunoelectrophoresis in agarose gel. On application of all these methods the lectin showed a differentiated metabolism, disappearing more slowly than the other high molecular weight proteins of the seed.     

Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus).

An acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.     

Ultrastructural detection of lectin receptors by cytochemical affinity reaction using mannan-iron complex.

A two-step affinity reaction is described for electron microscopic demonstration of the Concanavalin A as well as the Lens culinaris lectin receptors by means of the yeast mannan-iron complex. First the tissue was incubated in the lectin. Afterwards the incubation in the yeast mannan-iron complex was performed and reaction takes place between the still free second sugar binding site of membrane bound lectin molecules and the polysaccharides. This membrane receptor-lectin-polysaccharide complex is revealed by the electron dense iron core of the yeast mannan-iron complex. The specificity of the reactions could be demonstrated by addition of the hapten or by incubation in the yeast mannan-iron complex only. The proposed technique has proved useful for demonstration of lectin receptors in the small intestine.