Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

Isoiation and Characterization of Vicia FABA Lectin Affinity Purified on Chitin Column

Abstract

A lectin from early long pod var. of Vicia faba seed has been purified to homogeneity on chitin. The purified lectin is shown to be homogeneous in nature by Bio Gel P - 150 gel filtration, fast protein liquid chromatography and polyacrylamide gel electrophoresis. The lectin is a glycoprotein with molecular weight of 51,000. The lectin molecule is possibly composed of two types of subunits devoid of any covalent linking through sulfhydryl groups, with molecular weights 9,000 and 15,000 respectively in the ratio 2:2. The purified lectin shows a high affinity for N-acetyl-D-glucosamine (GlcNAc).

Amino acid analyses show that cysteine and methionine are absent, and a high proportion of aspartic acid and glutamic acid are present in the protein molecule. The extinction coefficient of the purified lectin is 7.22. The lectin behaves as a, cold agglutinin displaying stronger agglutination than the naturally occurring ABO agglutinin in the cold.     

Mucin binding mitogenic lectin from freshwater Indian gastropod Belamyia bengalensis: purification and molecular characterization

A lectin was purified from the hemolymph of the freshwater Indian gastropod Belamyia bengalensis. The purification involved successive ion-exchange chromatography on Resource Q and gel filtration on Superose 12 column in FPLC system. Homogeneity of the protein was confirmed by polyacrylamide gel electrophoresis. Belamyia bengalensis lectin (BBL) was a monomeric protein with a molecular weight of 33 kDa as demonstrated by gel filtration and SDS-PAGE. It is a glycoprotein containing 6% total sugar and its activity is highly dependent on Ca(2+). BBL agglutinated human erythrocytes and is a blood group non-specific lectin. It agglutinated animal erythrocytes also. Hapten inhibition studies indicated that BBL shows binding specificity only for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine at a high concentration among the mono- and oligosaccharides tested. Among the glycoproteins used for hemagglutination-inhibition assay, porcine submaxillary mucin was found to be the best inhibitor. Chemical modification studies indicated that Lys, Arg, and Trp are essential for the sugar-binding activity of BBL. Circular dichroism spectra revealed high content of alpha-helical structure in the lectin. BBL is a potent mitogen as it stimulated the T-lymphocyte proliferation, specifically the Th1 subset.     

Isolation and characterization of a lectin from tulip bulbs, Tulipa gesneriana

A lectin, which agglutinated specifically the yeast cells of the Saccharomyces genus, was isolated from tulip bulbs (Tulipa gesneriana) using affinity chromatography on mannan-Sepharose 4B. Its relative molecular mass was determined by gel filtration to be approximately 67,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a relative molecular mass of 17,000 was obtained, suggesting that the lectin is a tetramer. Binding studies performed with iodinated lectin indicated that Saccharomyces cerevisiae cells contained approximately 5.7 X 10(6) binding sites per cell, whereas little binding was observed with yeasts other than the Saccharomyces genus, bacteria and animal erythrocytes. D-Mannose, D-mannose 6-phosphate, L-fucose and L-fucosylamine were potent inhibitors of the lectin binding to S. cerevisiae cells, while, D-glucose, D-galactose and D-mannosamine were inactive, indicating that hydroxyl group at C-2 of D-mannose was essential for the lectin binding. Furthermore, inhibition experiments, using various manno-oligosaccharides, suggested that the lectin recognized (1----6)-linked manno-oligosaccharide units larger than mannobiose.     

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

Interaction of a rat intestinal brush border membrane glycoprotein with type-1 fimbriae of Salmonella typhimurium

Type-1 fimbriated Salmonella typhimurium was found to adhere to rat intestinal brush border membrane in a mannose sensitive manner. The maximum binding of the purified fimbriae observed with the rat illeal enterocytes was inhibited by 69.2% in presence of D-mannose. Brush border membrane from rat illeum was isolated, delipidified, solubilised and fractionated by affinity chromatography on type-1 fimbriae coupled Sepharose CL 4B column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the material eluted from the column with D-mannose revealed a single band of molecular weight 60 kDa. The direct binding of this affinity eluted glycoprotein to the purified type-1 fimbriae was demonstrated by a modified Western blot experiment. Our findings suggest that the 60 kDa glycoprotein may serve as a receptor for the type-1 fimbriae in the rat intestinal brush border membrane and thereby may help in mediating bacterial adherence to the host epithelium.     

Purification and characterization of a new lectin from the hard roe of skipjack tuna,Katsuwonus pelamis

Fish eggs are known as a rich source of lectins. In this study we purified and characterized a lectin from unfertilized Katsuwonus pelamis hard roe. K. pelamis lectin (KPL) was purified by separation into two fractions above and below the molecular weight of 10kDa using ultramembrane, gel filtration on a Sephadex G-100, and affinity chromatography on an asialofetuin-Sepharose 4B. KPL is a glycoprotein of 140kDa, composed mainly of aspartic acid, glycine, phenylalanine, glutamic acid, threonine and serine residues. Analysis of the carbohydrate composition by gas-liquid chromatography indicated that carbohydrates constituted 14% of the total weight and this 14% is comprised of mannose, galactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, fucose, arabinose and sialic acid. The lectin is comprised of four subunits. These subunits have a molecular mass corresponding to 35kDa. KPL specifically agglutinated human blood type A erythrocytes and, in a hemagglutination inhibitory test, the potent inhibitors were D-galactose, lactose, lactosamine, asialofetuin, N-acetyl-D-galactosamine, O-serinyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside and O-serinyl-2-acetamido-2-deoxy-beta-D-galactopyranoside (O-serinyl-beta-D-GalNAc). The first 10 residues of the N-terminal region were determined as PVELCDAKCT. Furthermore it was determined that the hemagglutinating activity of KPL was dependent on divalent metal cations and that the optimum activity of KPL was exhibited at 40 degrees C and pH 6.0-8.5 in the presence of Ca2+.     

Isolation and properties of a lectin from sainfoin (Onobrychis viciifolia, Scop.)

A glycoprotein capable of binding simple carbohydrates and causing hemagglutination has been isolated from seeds of the legume plant sainfoin (Onobrychis viciifolia, Scop. var Eski). The phytolectin was prepared by affinity chromatography of pH 7.0 sodium phosphate extracts on columns of Sepharose-4B containing covalently attached D-mannose. Molecular weight determinations showed the lectin to be a dimer consisting of 26 000 dalton, non-covalently associated monomers. Amino acid analyses indicated high amounts of aspartate, glutamate, threonine and serine which accounted for 41% of all amino acids. One residue of cysteine was present and methionine was totally absent. The lectin contained 2.6% (w/w) neutral carbohydrate and two residues of N-acetylglucosamine/monomer. Carbohydrate-binding specificity was directed toward D-mannose and D-glucose and their alpha-glycosidic derivatives. The purified protein agglutinated cat erythrocytes at 5 micrograms/ml. Antiserum to seed lectin showed a single common immunoprecipitation line in Ouchterlony double diffusion against both the seed and root antigen. Lectin isolated from sainfoin seedling roots showed molecular weight, amino acid and carbohydrate values similar to that of the seed lectin.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

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.     

A New Metabolite from Aspergillus versicolor

Neurospora sitophila produced extracellular and cell wall-associated lectins. The addition of l-sorbose to a culture resulted in a decrease in the production of the former lectin and complete abolition of the latter. The lectin in the culture filtrate was purified by bovine submaxillary mucin-conjugated Sepharose chromatography. The molecular weight of the lectin was calculated to be approx. 40,000 by Sephacryl S-200 gel filtration, and that of the subunit to be approx. 22,000 by SDS/polyacrylamide- gel electrophoresis. The lectin was not inhibited by simple sugars or their homopolymers. It was inhibited strongly by glycoproteins from human erythrocyte membrane and bovine submaxillary mucin, and moderately by α1-acid glycoprotein from human plasma, human IgA and IgM, and fetal calf fetuin. The lectin agglutinated human type A, B and O erythrocytes to the same degree. Erythrocytes from chick, horse, rabbit and sheep were more efficiently agglutinated.     

Isolation and characterization of a lectin from Annona muricata seeds

A lectin with a high affinity for glucose/mannose was isolated from Annona muricata seeds (Annonaceae) by gel filtration chromatography on Sephacryl S-200, ion exchange chromatography on a DEAE SP-5 PW column, and molecular exclusion on a Protein Pak Glass 300 SW column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) yielded two protein bands of approximately 14 kDa and 22 kDa. However, only one band was seen in native PAGE. The Mr of the lectin estimated by fast-performance liquid chromatography-gel filtration on Superdex 75 was 22 kDa. The lectin was a glycoprotein with 8% carbohydrate (neutral sugar) and required divalent metal cations (Ca2+, Mg2+, and Mn2+) for full activity. Amino acid analysis revealed a large content of Glx, Gly, Phe, and Lys. The lectin agglutinated dog, chicken, horse, goose, and human erythrocytes and inhibited the growth of the fungi Fusarium oxysporum, Fusarium solani, and Colletotrichum musae.     

中国鲎凝集素的分离纯化及性质研究

 经N-乙酰氨基葡萄糖交联琼脂糖亲和层析及以交联琼脂糖介质的高效液相分子筛层析,从中国鲎细胞溶解物中分离纯化了一种凝集素,其活性比原料鲎试剂提高128倍。鲎凝集素SDS电泳时表现出分子量为69000,和72000的二个亚基。N-乙酰氨基葡萄糖、D-半乳糖,D-甘露糖及岩藻糖等对鲎凝集素凝集鸡红细胞的活性有显著抑制作用,加热60℃,10分钟可使凝集素活性基本丧失。CaCl_2为凝集素活性所必需。鲎凝集素与肺炎球菌C多糖有沉淀反应。     

81 Recent introduction of polysiphonia morrowii (ceramiales, rhodophyta) to punta arenas, chile

In red algae, fertilization begins with gamete-gamete contact between the trichogyne cell wall of the female carpogonium and spermatial coverings. During the fertilization in Aglaothamnion oosumiense , reproductive cells interact with each other through sex specific adhesion molecules on the surface of spermatia and trichogyne. The gamete binding is highly selective suggesting the presence of recognition factors along their surfaces. In the previous studies, we have reported that spermatial binding to trichogynes of a red alga, Aglaothamnion oosumiense is mediated by a lectin-carbohydrate complementary system. Spermatial binding to trichogynes was inhibited by pre-incubation of trichogynes with N-acetyl-D-galactosamine and D-glucose and hence lectins specific to these sugars were expected to present on the surfaces of trichogyne cell wall. We have isolated a new lectin from Aglaothamnion oosumiense by the use of agarose bound N-acetyl-D-galactosamine affinity chromatography and named it as rhodobindin. Rhodobindin agglutinated human erythrocytes as well as spermatia of Aglaothmanion oosumiense . The agglutinating activity of this lectin was inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. SDS-PAGE results showed that this lectin may be monomeric. The molecular weight was determined as 21,876 dalton by matrix-assisted laser desorption ionization (MALDI) mass-spectrometry. N-terminal amino acid sequence of the lectin was analyzed and revealed to have no identity with those of known proteins. The complementary male glycoprotein was also isolated and purified by the use of SBA-agarose affinity chromatography. The subtractive cloning was carried out to characterize the recognition molecules.     

A chitotetrose specific lectin fromLuffa acutangula: Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ±1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S₂₀, w) was obtained to be 4.06 S. The Stokes’ radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.     

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.     

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.     

A barley lectin that binds free amino sugars I. Purification and characterization

A lectin was isolated from barley seed which bound the coat glycoprotein of barley stripe mosaic virus (Type strain) and precipitated the virus from solution. Purification of the barley lectin was achieved by fractionation with ammonium sulfate and successive column chromatography on DEAE cellulose and cellulose phosphate. The barley lectin was homogeneous as ascertained by polyacrylamide gel electrophoresis, isoelectric focusing, and from immunochemical tests. No isolectins were detected. The lectin has a molecular weight of 31 000 daltons and is not a glycoprotein. Each virion can accomodate between 200 to 300 molecules of lectin. Barley lectin was shown to be specific for D-glucosamine, D-galactosamine and D-mannosamine with little distinction among the epimeric configurations at carbons 2 and 4. Free amino groups of D-glucosamine and D-galactosamine were detected on the coat glycoprotein of Type strain barley stripe mosaic virus and these sugars appear to serve as receptors for the barley lectin.     

Secretion of a lactosaminoglycan-containing glycoprotein by peri-implantation sheep conceptuses

Sheep conceptuses from day 16 of pregnancy were cultured in the presence of [3H]glucosamine and [14C]leucine and a high-molecular-weight glycoprotein (HMWG) secreted into the culture medium was purified by a combination of anion-exchange and gel filtration chromatography. The HMWG was found to have a molecular weight between 800,000 and 900,000 and to be highly resistant to digestion with pronase. Characteristics of the carbohydrate portion of the purified glycoprotein were examined by selective chemical and enzymatic digestions and lectin binding studies. Mild alkaline reduction was ineffective in disassociating carbohydrate chains from the protein core. Furthermore, the protein was resistant to both O-glycanase and peptide:N-glycanase F. Harsh alkaline reduction caused the release of carbohydrates, however. After pronase digestion of these products, three molecular weight classes of carbohydrates were resolved by Sephadex G-25 chromatography. Two lines of evidence indicate that the HMWG contains lactosaminoglycan components. The intact molecule and two of the molecular weight classes of carbohydrates resolved by harsh alkaline reduction bind Datura stramonium lectin. Binding of HMWG to lectin could be partially inhibited by N-acetyllactosamine and completely inhibited by a mixture of N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. Secondly, digestion with endo-beta-galactosidase causes the release of 16% of the [3H]glucosamine from the intact molecule. Therefore, the HMWG of the sheep conceptus is the first reported example of secretion of lactosaminoglycan-containing glycoprotein by peri-implantation embryos.     

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.