Production and characterization of lectin from <Emphasis Type="Italic">Bauhinia holophylla</Emphasis> (Fabaceae:Cercideae) calli

Bauhinia holophylla is a woody plant found in the Brazilian Cerrado used in folk medicine to treat diabetes. Lectins are glycoproteins with several biotechnological applications and play important roles in plant defenses and in medicine. Lectins have been produced in vitro using plant tissue culture techniques. This study shows the production and characterization of lectin from B. holophylla by callus culture. Calli were obtained from leaf segments placed on woody plant and Murashige and Skoog media supplemented with 2,4-dichlorophenoxyacetic acid (0, 4.52, 9.05, 18.10 µM) or 6-benzylaminopurine (0, 4.44, 8.88, 17.75 µM), in the presence or absence of light. The highest concentrations of lectin expressed by hemagglutination activity were observed in green and compact callus induced in woody plant medium supplemented with 6-benzylaminopurine in the presence of light, which showed specificity by all types of erythrocytes tested. The highest concentrations of lectin (128 HU mg^?1) and fresh weight of callus were observed in the stationary phase (41st–60th day). Lectin was not detected in subcultured calli. d-Galactose promoted the highest inhibitory effect on the lectin activity in human erythrocytes ABO system, especially B-type. Lectin has been shown to be a stable protein, maintaining its hemagglutination activity after incubation at a wide range of temperatures, pH values and ethylenediaminetetraacetic acid concentrations.     

紫藤凝集素的分离纯化及理化性质研究

用常规方法处理的ＤＥＡＥ离子交换纤维素柱,通过线性离子强度梯度洗脱,从紫藤种子的蛋白粗提液中得到一定纯度的紫藤凝集素。纯化的凝集素凝集兔红血球的比活提高４０倍,总活力回收率为１９．２％。紫藤凝集素的分子量经ＰＡＧＥ鉴定为２０５ｋｄ,是由两种亚基构成的四聚体,这两种亚基各有２个,分子量ＳＤＳ－ＰＡＧＥ鉴定分别为７７６００ｄ和２５１００ｄ。紫藤凝集素是一种糖蛋白,等电点约为４．６０。它可凝集人的各种血     

Heparin-binding lectin from human placenta: further characterization of ligand binding and structural properties and its relationship to histones and heparin-binding growth factors

We have previously demonstrated that the heparin-binding lectin of human placenta dissociates into up to four distinct polypeptides with molecular weights of 14,400, 15,000, 16,200, and 16,700 (Kohnke-Godt, B., & Gabius, H.-J. (1989) Biochemistry 28, 6531-6538). Stable complexes to ligands can shift the molecular weight appearance of the lectin to higher values. They can be dissociated in the additional presence of 9 M urea or by enzymatic degradation of heparin in model studies. The binding of heparin is rather stable over a range of salt concentrations from 1 to 3 M NaCl. Chemical modification with group-specific reagents to arginine, lysine, histidine, tyrosine, and tryptophan results in substantial inactivation of binding activity. Further amino-terminal sequence analyses point to a high-scoring relationship in this region to histone sequences, namely, histone H2B, but to no published sequences for any heparin-binding growth factor. Calculation of relatedness on the basis of differences in amino acid composition corroborates the conclusion of molecular distinction between the lectin, histones H2A and H2B, and the fibroblast growth factor as well as angiogenin. Histones only weakly agglutinate type II erythrocytes in contrast to the lectin. The immobilized lectin exhibits two classes of binding sites with KD values of 3 and 110 nM in contrast to one estimated KD value of 250 nM with a commercially available histone fraction. Both fractions retain binding activity to biotinylated heparin in transblots and are immunologically cross-reactive to antibodies, raised against the lectin as antigen. Subcellular fractionation clearly demonstrates that heparin-inhibitable hemagglutination activity and immunologically cross-reactive protein bands, characteristic for the lectin, but not unequivocally distinguishable from certain histone fractions in blots, are not confined to the nuclear fraction in the human placenta.     

Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra)

A novel lectin was purified from the coelomic fluid of the sea cucumber Holothuria scabra (HSL), subjected to bacterial challenge. HSL is a monomeric glycoprotein of molecular mass 182 kDa. The lectin is highly thermostable as it retains full activity for 1 h at 80 degrees C. Further, the hemagglutination activity of HSL is unaffected by pH in the range 2-11. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HSL does not require any divalent metal ions. The affinity profile of HSL was studied by a combination of hemagglutination inhibition and fluorescence spectroscopy. HSL binds to desialylated glycoproteins, MealphaGal, T-antigen and T (alpha-ser)-antigen with a distinction between beta1-4 and beta1-3 linkages. Mealpha-T-antigen was a potent ligand having highest affinity (Ka 8.32 x 10(7)M(-1)). Monosaccharide binding is enthalphically driven while disaccharide binding involves both entropic and enthalpic contributions.     

A simple procedure for the isolation of l-fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus

l-Fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus were isolated by affinity chromatography on columns of l-fucose-Sepharose 6B. l-Fucose was coupled to Sepharose 6B after divinyl sulfone-activation of the gel to give an affinity adsorbent capable of binding more than 1.2 mg of Ulex lectin/ml of gel, which could then be eluted with 0.1M or 0.05M l-fucose. Analysis of the isolated lectins by hemagglutination assay, by gel filtration, and by polyacrylamide discelectrophoresis revealed the presence of isolectins, or aggregated species, or both. The apparent mol. wt. of the major lectin fraction from Lotus was 35 000 when determined on Sephadex G-200 or Ultrogel AcA 34. In contrast, the apparent mol. wt. of the major lectin fraction from Ulex was 68 000 when chromatographed on Sephadex G-200 and 45 000 when chromatographed on Ultrogel AcA 34. The yields of lectins were 4.5 mg/100 g of Ulex seeds and 394 mg/100 g of Lotus seeds.     

Heparin-inhibitable lectins: Marked similarities in chicken and rat

Extracts of young rat lung contain a heparin-inhibitable lectin that closely resembles one recently purified from chicken liver. Both lectins interact with heparin and N-acetyl-D-galactosamine, and were purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. They both behave as high molecular weight aggregates that can be dissociated into two peptides with apparent molecular weights of 13,000 and 16,000 by gel electrophoresis in SDS. Samples of purified lectin contained up to 20% DNA by weight, and the degree of lectin aggregation and hemagglutination activity was greatly reduced by treatment with micrococcal nuclease without inhibiting heparin-binding activity. Association of lectin with DNA is an artifact of homogenization in high salt, since only 2% of the lectin is found associated with a purified nuclear fraction.     

Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.     

Changes in the level of lectins in the mantle of the mussel Mytilus trossulus in response to anthropogenic contaminants

The lectin activity in an extract from the mantle of the mussel Mytilus trossulus was tested for the first time. Using the method of the hemagglutination inhibition assay, it was shown that lectins were Gal/GalNAc-specific and best agglutinated with rabbit erythrocytes. The influence of foreign compounds on the lectin level in the M. trossulus mantle was examined. Upon cadmium acetate exposure, the level of lectin activity exhibited phasic alterations and depended on the contaminant concentration or the time of exposure. During exposure of mussels to a synthetic detergent or diesel fuel, changes in lectin contents were dependent on the time of exposure.     

Lectins from seeds of Crotalaria pallida (smooth rattlebox)

A lectin from the seeds of Crotalaria pallida (CPL), with an apparent molecular mass of 30 kDa, determined by SDS-polyacrylamide gel electrophoresis, showed human type A and B erythrocytes agglutination activity, which is inhibited by raffinose and galactose. The lectin requirement for divalent cation was demonstrated with EDTA/EGTA blocking hemagglutination activity. Although the N-terminal amino acid sequence of CPL is identical to another lectin from Crotalaria striata, which is taxonomically synonymous to Crotalaria pallida, these lectins differ in amino acid composition and hemagglutination properties.     

Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.     

Distribution of Lectins in Tissues, Derived Callus, and Roots of Psophocarpus tetragonolobus (Winged Bean)

The distribution of lectin in parental tissues, roots formed de novo from parental stem tissue, and derived callus cells of Psophocarpus tetragonolobus has been measured by hemagglutinating activity and radioimmunoassay. The antisera used for the radioimmunoassay was raised in rabbits to lectin isolated from seeds by affinity chromatography using insolubilized hog gastric mucin. The distribution of lectin in buffer extracts of the tissues (or cells) and the extracellular medium favors the tissues for in vitro grown roots, regardless of the culture conditions used. The lectin content of the extracellular medium is more significant for callus, regardless of its conditions of culture. The lectin activity of extracts of in vitro grown roots was higher than that of mature roots from whole plants. Differences in relative levels of lectin activity measured by hemagglutination and by radioimmunoassay, and differences in saccharide inhibition of hemagglutination, suggest the presence of multiple lectins in extracts of different tissues.     

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.     

Change of the wheat lectin activity and degree of its interaction with different components of compositions of lectin nature

The wheat lectin hemagglutination activity and degree of its interaction with the bacterium Azotobacter chroococcum T79 and aminosaccharide N-acetyl-D-glucosamin hapten of wheat lectin was studied in laboratory experiments with the purpose of creation of biologic activity compositions of lectin nature for plant growing. It was shown that plant-bacterial compositions encloses the "bacteria+lectin" complex, free lectin and bacterial cells. The addition of aminosaccharide N-acetyl-D-glucosamin to wheat lectin, to the bacterial culture and plant-bacterial composition decreases its hemagglutination activity. The possibility of creation of new complexes in this compositions effected by hapten "lectin+hapten", "lectin+hapten+bacteria", "bacteria+hapten" is under discussion.     

Amino acid sequence and molecular characterization of a D-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs

The complete amino acid sequence of a 11.5-kDa subunit of D-galactoside binding lectin purified from sea urchin (Anthocidaris crassispina) eggs is presented. The 105-residue sequence of the subunit was determined by analysis of the intact S-carbamoylmethylated protein and peptides generated by digestion with Achromobacter protease I or Staphylococcus aureus V8 protease. The lectin exists as a disulfide-linked homodimer of two subunits; the dimeric form is essential for hemagglutination activity. However, the monomeric form obtained by partial reduction retains the carbohydrate binding capacity. Neither Ca2+ nor SH reagent is essential for hemagglutination or carbohydrate binding. The sequence has no similarity to that of any known protein and apparently represents a new type of galactoside binding lectin.     

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.     

High molecular weight lectin isolated from the mucus of the giant African snail Achatina fulica

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca(2+)-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

Isolation and characterization of a lectin from the seeds of Dioclea lehmanni.

Affinity chromatography of the globulin fraction from the seeds of Dioclea lehmanni on Sephacryl S-200 yielded two lectins, one slightly retarded and another strongly bound. The latter, which was a glucose/mannose specific lectin, was purified and the following properties were determined: pI, Mr of subunits, carbohydrate content, A, aminoacid composition, hemagglutination and inhibition patterns, N-terminal sequence and mitogenic activity. These properties of the lectin were very similar to those of the Con A and Dioclea grandiflora lectins.     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the 'microsomes' (13000-80000 X g sediment) from pea stem tissue is strongly influenced by concentration of Mg2+ in the homogenization medium. The absence of Mg2+ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at 80000 X g upon addition of 5-10 mM Mg2+ (or Mn2+, Ca2+, Zn2+) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl2 the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg2+. Microsomes prepared with Mg2+ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Affinity purification and characterization of a seed lectin from Crotalaria medicaginea

Purification of lectin from the seeds of Crotalaria medicaginea Lamk by affinity chromatography on asialofetuin-linked amino activated silica, yielded a single band on non-denatured PAGE at pH 4.5 and 8.3 and, a single peak on HPLC size exclusion and cation exchange columns. The molecular mass of the native C. medicaginea lectin was determined to be 125 kDa by gel filtration. In SDS-PAGE, the lectin migrated as a single band of M(r) 31.6 kDa under reducing and nonreducing conditions, indicating that it is a tetramer of apparently identical subunits. It agglutinated red blood cells (RBCs) from rabbit and human ABO blood groups. It also reacted with RBCs from rat, sheep, goat and guinea pig but after desialylation with neuraminidase. The hemagglutination activity of the lectin was inhibited by D-galactose and its derivatives. Amino acid analysis showed that lectin was rich in aspartic and glutamic acid and, did not contain sulphur containing amino acids. The lectin is a glycoprotein having 1.41% of neutral sugars. It is labile at temperature above 60 degrees C. It needs divalent cations for its activity, as a loss of activity was observed on removal of Ca2+ and Mn2+. Denaturing agents like urea, thiourea and guanidine-HCl have no effect on its activity.