Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

A lectin from Bryonia dioica root stocks

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.Abbreviations BDA Bryonia dioica agglutinin - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

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

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

     

Xanthosoma sagittifolium tubers contain a lectin with two different types of carbohydrate-binding sites

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Galbeta1,4GlcNAc-) or lacto-N-biose (i.e. Galbeta1,3GlcNAc-) groups at its three nonreducing termini.     

Purification and properties of lung lectin. Rat lung and human lung beta-galactoside-binding proteins.

Lung is one of the organs of the rat with a particular abundance of haemagglutinating activity that is inhibited by beta-galactosides. This lectin activity can be attributed to a single protein that has been purified from rat lung; a similar protein has been purified from human lung. The molecular weights and subunit structures were estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the human lung lectin appeared to be composed to two identical subunits, mol.wt. 14500, whereas rat lung lectin was observed as both a dimer and a tetramer of one subunit type, mol.wt. 13500. Both lectins bind to disaccharides or oligosaccharides with terminal beta-linked galactose residues. The carbohydrate moiety may be free [lactose or D-galactopyranosyl-beta-(1 leads to 4)-thiogalactopyranoside], protein-bound (asialofetuin) or lipid-bound (cerebrosides). The molecular properties of the beta-galactoside-binding proteins of rat lung and human lung are closely similar to those of embryonic chick muscle lectin [Nowak, Kobiler, Roel & Barondes (1977) Proc. Natl. Acad. Sci. U.S.A. 73, 1383--1387] and calf heart lectin [De Waard, Hickman & Kornfeld (1976) J. Biol. Chem. 251, 7581--7587].     

Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Purification and characterization of a human lectin specific for penultimate galactose residues

A novel lectin has been found in human plasma. The lectin was purified by affinity chromatography using an adsorbent in which 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosylhydroxylysine (Glc-Gal-Hyl) was coupled to Sepharose. The molecular weight of the lectin was determined by gradient gel electrophoresis to be approximately 240,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the subunit had the molecular weight of 29,500. Composition analysis has shown the lectin is a glycoprotein in which 12% of the molecule consists of carbohydrate. Native human, horse, calf, sheep, rabbit, and rat erythrocytes were agglutinated by the lectin in the presence of calcium. Glc-Gal-Hyl, N-acetylated Glc-Gal-Hyl, and stachyose inhibited the hemagglutination, whereas monosaccharides, maltose, cellobiose, lactose, raffinose, galactosylhydroxylysine, and N-acetylated galactosylhydroxylysine were not inhibitory. The lectin is strongly inhibited by the desialylated bovine erythrocyte glycoprotein, which contains galactose beta 1-3galactose beta-sequence at the nonreducing termini of the sugar chains, whereas disialylated orosomucoid did not inhibit the lectin. These results indicate that the lectin recognizes the penultimate galactose residue in a hapten molecule in contrast to usual galactose-binding proteins or galactose-specific lectins, which recognize exposed, terminal galactose residues of sugar chains.     

Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.     

Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.     

Microheterogeneity in purified broad bean polyphenol oxidase

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5.     

An anti-A1 lectin in the seeds of Amphicarpaea bracteata

An anti-A1 lectin has been isolated from the extract of Amphicarpaea bracteata seeds by affinity chromatography on Epoxy-activated Sepharose 6B coupled to N-acetyl-D-galactosamine. The yield of the purified lectin was 86 microgram/g of seeds. The purified lectin shows one main band on electrophoresis in sodium dodecyl sulfate-polyacrylamide. The amino acid and neutral sugar composition indicate that this lectin is an acidic glycoprotein with a neutral sugar content of approx. 2%. The composition of the lectin is different from that of the Dolichos biflorus lectin but the two lectins have some common characteristics. The most powerful inhibitors of the agglutination of A1 red blood cells by the A. bracteata lectin is N-acetyl-D-galactosamine. Much weaker inhibitors of the agglutination are alpha-lactose, D-fucose, and five other sugars.     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

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.     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.