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.     

Isolation and properties of a lectin from the seeds of the Indian bean or lablab (Dolichos lablab L.).

The lectin of the Indian bean or lablab (Dolichos lablab L.) was purified by affinity chromatography on two types of affinity carriers: O-alpha-D-mannopyranosyl-Separon and Separon-bound ovomucoid. The lectin is homogeneous in the ultracentrifuge: S20, w = 6.14 S, Mr = 110 000; the molecule appears to comprise two pairs of two types of subunits (Mr 16 000 and 40 000), and contains 2% neutral sugar and 0.2 Mn and 0.5 Zn atom respectively. The lectin agglutinates human erythrocytes non-specifically with regard to ABO grouping at a limit concentration of 8 micrograms/ml, and this activity is inhibited most effectively by N-acetyl-D-glucosamine, methyl alpha-D-mannopyranoside and ovomucoid, but not by free D-mannose.     

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.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

A new lectin from the sea worm Serpula vermicularis: isolation, characterization and anti-HIV activity

A GlcNAc-specific lectin was isolated from the sea worm Serpula vermicularis (SVL) (Annelida) and purified by ion-exchange, affinity and gel permeation chromatography. SVL was a homotetrameric protein with native molecular mass of about 50 kDa, and consisted of identical subunits of 12.7 kDa. The carbohydrate content of 1.9% suggested that the lectin was a glycoprotein, and mainly composed by aspartic and glutamic acids, glycine, valine and serine; with relatively lower content of basic amino acids and cysteine. The first 15 residues of the N-terminal region were determined as ADTPCQMLGSRYGWR. It was stable at pH 6-9 and at temperatures up to 40 degrees C. SVL was Ca(2+)-independent lectin that agglutinated native and trypsinized human erythrocytes. Hapten inhibition studies indicated that SVL showed binding specificity only for N-acetyl-d-glucosamine and its derivatives among the monosaccharides tested and required the presence of hydroxyl group at the C-3 of GlcNAc. The presence of hydrophobic p-nitrophenyl aglycone improved inhibitory potency of N-acetyl-d-glucosamine. Ovomucoid and ovalbumin were found to be inhibitors among the glycoproteins used for inhibition assay. The anti-HIV-1 (human immunodeficiency virus) activity of SVL in vitro was determined: SVL inhibited the production of viral p24 antigen and cytopathic effect induced by HIV-1. The EC(50) values were 0.23 and 0.15 microg x mL(-1) respectively.     

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.     

Affinity purification, physicochemical and immunological characterization of a galactose-specific lectin from the seeds of Dolichos lablab (Indian lablab beans)

A galactose-specific lectin has earlier been isolated from the seeds of Dolichos lablab in our laboratory by conventional protein purification methods. We now established conditions to bind the lectin on Sepharose-galactose gel in the presence of 1.5 M ammonium sulfate in Tris-buffered saline, pH 7.4. It can be specifically eluted with 0.3 M galactose. The purified lectin is a glycoprotein, binds to Con A, agglutinates erythrocytes, and has an apparent native molecular weight of 120 +/- 5 kDa. In SDS-PAGE under reducing conditions, it dissociates into two subunits of molecular mass (Mr) 31 and 29 kDa. Among a number of sugars tested for inhibitory activity of the lectin, galactose was found to be a potent inhibitor. Rabbit polyclonal antibody to the purified lectin specifically reacted with the lectin subunits in Western blot analysis and additionally, an antibody raised to the isolated 31 kDa subunit show reactivity with both the subunits. Amino terminal sequences of both the subunits are identical. The purified lectin is stable up to 40 degrees C with a pH optimum of 7.4. The lectin has a high content of acidic amino acids and lacks sulfur-containing amino acids. Chemical modification of the lectin with group-specific reagents indicates the possible role of histidine, lysine, and tyrosine residues in lectin activity.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a second lectin (SNA-II) present in elderberry (Sambucus nigra L.) bark

A second lectin (SNA-II) has been isolated from elderberry (Sambucus nigra L.) bark by affinity chromatography on immobilized asialo-glycophorin. This lectin is a blood group nonspecific glycoprotein containing 7.8% carbohydrate and which is rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, valine, and leucine. Gel filtration on Superose 12 gave a single symmetrical peak corresponding to Mr, 51,000; SDS-acrylamide electrophoresis gave a single polypeptide, Mr, 30,000. Hence SNA-II appears to be a homodimer. The lectin is a Gal/GalNAc-specific lectin which is precipitated by glycoproteins containing GalNAc-terminated oligosaccharide chains (e.g., asialo-ovine submaxillary and hog gastric mucins), and by glycoproteins and polysaccharides having multiple terminal nonreducing D-galactosyl groups as occur in asialoglycophorin, asialo-laminin and Type 14 pneumococcal polysaccharide. The carbohydrate binding specificity of SNA-II was studied by sugar hapten inhibition of the asialo-glycophorin precipitation reaction. The lectin's binding site appears to be most complementary to Gal-NAc linked alpha to the C-2, C-3, or C-6 hydroxyl group of galactose. These disaccharide units are approximately 100 times more potent than melibiose, 60 times more potent than N-acetyllactosamine, and 30 times more potent than lactose. Interestingly, the blood group A-active trisaccharide containing an L-fucosyl group linked alpha 1-2 to galactose was 10-fold poorer as an inhibitor than the parent oligosaccharide (GalNAc alpha 1-3Gal), suggesting steric hindrance to binding by the alpha-L-fucosyl group; this explains the failure of the lectin to exhibit blood group A specificity.     

Chemical modification studies on the glucose/mannose specific lectins from field and lablab beans.

Seeds of Dolichos lablab var. lignosus (field beans) and variety typicus (lablab beans) contain glucose/mannose specific lectins that have been affinity purified and well characterised (Siva Kumar N., and Rajagopal Rao, D., J.Biosci., 1986, 10, 95-109, (1) Rajasekhar et al., (Biochem.Archives. 1997, 13, 233-240) (2). Purified lectins are glycoproteins with a native molecular mass of 60 kDa and are made of two types of subunits (Gowda et al., 1994, J.Biol.Chem. 269, 18789-18793) (3). Chemical modifications of various groups in purified lectins (using group specific reagents) such as lysine (citraconic anhydride), carboxyl groups (water soluble carbodiimide) tyrosine (N-acetyl imidazole) and tryptophan (2-hydroxy 5-nitro benzylbromide) revealed that 14 out of 21 residues of lysines 7 out of 92 residues of carboxyl groups, 16 out of 24 tyrosine residues and 2 out of 10 tryptophan residues were modified. Lysine and carboxyl group modification led to 95% loss in haemaglutinating activity compared to control while tyrosine and tryptophan modifications led to complete loss of lectin activity. Arginine and histidine modifications led to only 50% loss in activity. The extent of modification and loss in activity was same when the lysine and carboxyl groups were modified in the presence and absence of the inhibitory sugar methyl alpha-D-glucopyranoside at 0.1 M concentration. However protection of modification and lectin activity was observed when the tyrosine and tryptophan residues were modified in the presence of the inhibitory sugar. Earlier CD studies carried out (1) and extensive chemical modification studies reported here substantiate the involvement of tyrosine and tryptophan residues in the sugar binding site of these lectins.     

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.     

Insulin-like growth factor-binding protein from human plasma. Purification and characterization

Insulin-like growth factor (IGF)-binding protein (BP) has been purified from Cohn fraction IV of human plasma by acidification, ion exchange to remove endogenous ligands, and affinity chromatography on agarose-IGF-II. The pure protein appeared as a single peak by high performance reverse-phase and gel permeation chromatography (molecular mass, 45-50 kDa), but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band at 53 kDa and a minor band at 47 kDa, unreduced, or 43 and 40 kDa, respectively, reduced. The two bands stained for both protein and carbohydrate. After storage at 2 degrees C for 5 months at pH 3, two additional bands, at 26 and 22 kDa on unreduced gels, were also present. Autoradiography after affinity labeling with IGF-I or IGF-II tracer revealed a single labeled band of 61 kDa. BP, quantitated using a specific radioimmunoassay, was retained by agarose-immobilized IGF-I, IGF-II, concanavalin A, and wheat germ lectin, but not Helix pomatia lectin. Competitive binding curves using pure BP and human IGF-I and IGF-II as both labeled and unlabeled ligands indicated association constants of 2-3 X 10(10) liters/mol for both peptides, with a slightly higher affinity for IGF-II than IGF-I, and 0.9 binding sites for either peptide per 53-kDa protein. The exact relationship of this acid-stable IGF BP to the 150-kDa complex from which it is derived remains to be determined.     

Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.     

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean)

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I > Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.     

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 tuber lectin from Arisaema jacquemontii Blume with anti-insect and anti-proliferative properties

A tuber lectin from Arisaema jacquemontii Blume belonging to family Araceae was purified by employing a single step affinity chromatography using column of asialofetuin-linked amino activated silica beads and the bound lectin was eluted with 100 mM glycine-HCl buffer pH 2.5. The purified A. jacquemontii lectin (AJL) showed a single protein band with an apparent molecular mass of 13.4 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The native molecular mass of AJL determined by gel filtration on a Biogel P-200 column was 52 kDa and its carbohydrate content was estimated to be 3.40%. Thus AJL is a tetrameric glycoprotein. The purified lectin agglutinated erythrocytes from rabbit but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested except N-acetyl-D-lactosamine having minimal inhibitory sugar concentration (MIC) 25 mM. Among the glycoproteins tested only asialofetuin was found to be inhibitory (MIC125 microg/mL). A single band was obtained in native PAGE at pH 4.5 while PAGE at pH 8.3 showed two bands. Isoelectric focusing of AJL gave multiple bands in the pI range of 4.6-5.5. When incorporated in artificial diet AJL significantly affected the development of Bactrocera cucurbitae (Coquillett) larvae indicating the possibility of using this lectin in a biotechnological strategy for insect management of cucurbits. Larvae fed on artificial diet containing sublethal dose of AJL showed a significant decrease in acid phosphatase and alkaline phosphatase activity while esterase activity markedly increased as compared to larvae fed on diet without lectin. Out of various human cancer cell lines employed in sulphorhodamine B (SRB) assay, this lectin was found to have appreciable inhibitory effect on the in vitro proliferation of HCT-15, HOP-62, SW-620, HT-29, IMR-32, SKOV-3, Colo-205, PC-3, HEP-2 and A-549 cancer cell lines by 82, 77, 73, 70, 41, 41, 37, 29, 21 and 21% respectively.