Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

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.     

A putative carbohydrate-binding domain of the lactosebindingCytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of thel-fucose-bindingUlex europaeus anti-H(O) lectin

The complete amino acid sequence of a lactose-bindingCytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of thel-fucose-bindingUlex europaeus lectin I (UEA-I).Abbreviations BPA Bauhinia purpurea lectin - Con A concanavalin A - CMA-I Cytisus multiflorus lectin I - CMA-II Cytisus multiflorus lectin II - CSA-I Cytisus sessilifolius lectin I - CSA-II Cytisus sessilifolius lectin II - CSII Cytisus scoparius lectin II - ECorL Erythrina corallodendron lectin - GSIV Griffonia simplicifolia lectin IV - HPLC high performance liquid chromatography - LAA-I Laburnum alpinum lectin I - LAA-II Laburnum alpinum lectin II - LOL Lathyrus ochrus lectin - LTA Lotus tetragonolobus lectin - MAH Maackia amurensis haemagglutinin - PSA Pisum sativum lectin - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid - UEA-I Ulex europaeus lectin I - UEA-II Ulex europaeus lectin II - VFA Vicia faba lectin     

The primary structure of the Cytisus scoparius seed lectin and a carbohydrate-binding peptide.

The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

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.     

Purification and properties of two lectins from the latex of the euphorbiaceous plants Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge).

1. From the latex of two members of the plant family Euphorbiaceae, Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge), two lectins were purified by affinity chromatography on acid-treated Sepharose 6B followed by elution with D-galactose. 2. The lectin from E. characias is a single molecular species with Mr 80 000, made up of two identical subunits with Mr 40 000, and is a glycoprotein containing 11% carbohydrate. 3. The lectin from H. creptians appears as a mixture of three isolectins with Mr 140 000, consisting of four different subunits with Mr values 37 500, 35 500, 31 000, and 29 000. 4. Both lectins have haemagglutinating activity, with no specificity for human blood groups. The haemagglutinating activity is inhibited by D-galactose and by galactose-containing oligosaccharides. 5. The lectin from H. crepitans is mitogenic to human T-, but not to B-, lymphocytes. The latex of E. characias is mitogenic to T- and, to a lesser extent, to B-, lymphocytes, but the purified E. characias lectin has no mitogenic activity. 6. The lectin from H. crepitans, but not that from E. characias, inhibits protein synthesis by a rabbit reticulocyte lysate.     

Human erythrocyte specific lectin from the seeds of Indian coral tree,<Emphasis Type="Italic">Erythrina variegata</Emphasis> Linn,var.<Emphasis Type="Italic">orientali</Emphasis> Linn,Merrill

Human erythrocyte specific lectin was isolated from the seeds ofErythrina variegata Linn. var.orientalis Linn. Merrill. The lectin preferentially agglutinated erythrocytes in the sequence of O>B>A = AB. The lectin was purified 19-fold by affinity chromatography on acid treated sepharose 4B with an yield of 81%. The purified lectin was found homogeneous on polyacrylamide gel electrophoresis. The erythroagglutination reaction was inhibited by N-acetyl-D-galactosamine, D-galactose and lactose at very low concentration. The haemagglutination by the purified lectin was not inhibited by different hexose and pentose sugars even at high concentration. The purified lectin was a glycoprotein and agglutinated leucocytes at 3 μg protein concentration. The lectin induced transformation of peripheral blood lymphocytes in cultures.     

Galactose-particle receptor on liver macrophages. Quantitation of particle uptake

Liver macrophages have been shown previously to bind and ingest gold particles coated with asialoglycoproteins via a N-acetyl-D-galactosamine / D-galactose-specific lectin (Kolb-Bachofen, V., Schlepper-Sch?fer, J., Vogell, W. and Kolb, H. (1982) Cell 29, 859-866). We present here a quantitative analysis of lectin-dependent particle endocytosis. We used a conjugate of asialofetuin with colloidal gold as ligand, the cellular uptake of which could be followed by spectrophotometry. Freshly isolated Kupffer cells from the rat liver ingest asialofetuin at a rate of approx. 4200 particles/cell per min. Uptake is inhibited by saccharides related in structure to D-galactose and depends on the presence of Ca2+. The rate of endocytosis is zero below 10 degrees C, shows a modest increase until 20 degrees C and a steep increase between 20 and 37 degrees C. Uptake is energy-dependent and strongly inhibited by cytochalasin B but only slightly by colchicine.     

Isolation and characterization of a lectin from the roots of Dolichos biflorus

A lectin has been isolated from the roots of 7-day-old Dolichos biflorus plants and has been compared with the D. biflorus seed lectin. The root lectin differs from the seed lectin in molecular weight, subunit stoichiometry, amino acid composition, amino terminal amino acid sequence, and isoelectric focusing pattern. However, the root lectin has in common with the seed lectin a specificity for N-acetyl-D-galactosamine, and upon denaturation the root lectin will react weakly with antiserum made to denatured seed lectin. Distribution studies of this lectin in germinating seedlings show that the highest levels of lectin are found in 1-day-old roots. Upon dissection and analysis of 7-day-old roots, the highest levels of the lectin are in the uppermost segment. In addition, isoforms of this lectin also exist in the stems and leaves of the plant.     

Blood group B-specific lectin of Plecoglossus altivelis (Ayu fish) eggs

A lectin that agglutinates human blood group B erythrocytes but not blood group A and O erythrocytes was isolated from eggs of Ayu sweet fish (Plecoglossus altivelis). The lectin also agglutinates Ehrlich ascites carcinoma cells but not rat ascites hepatoma AH109 or rat sarcoma 150 cells tested. The lectin agglutination was most effectively inhibited by monosaccharides with the first type of configuration, i.e., L-rhamnose, L-mannose and L-lyxose at a concentration of 0.03 mM. The lectin agglutination was moderately inhibited by monosaccharides with the second type of configuration, i.e., D-galactose, D-fucose and D-galacturonic acid at a concentration of 0.4 mM. However, the agglutination was not inhibited by various other monosaccharides and oligosaccharides that have other types of configuration. The basis for an apparent B-specific hemagglutination may be due to the steric similarity of the C2 and C4 of the galactosyl series, the B-specific determinant, and the L-rhamnosyl-Sepharose column and was characterized as a homogeneous low molecular weight protein (Mr 14000) with an abundance of hydrophobic amino acids and dicarboxylic amino acid.     

A new mitogenic D-galactosephilic lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins

The lectin of Erythrina corallodendron (Caesalpiniaceae) seeds was purified by heating, ammonium sulfate fractionation, and affinity chromatography on acid-treated Sepharose. The purified lectin is similar to the soybean lectin in being a glycoprotein of molecular weight around 110 000 - 120 000 and having D-galactosephilic activity. This lectin, like the soybean and Pseudomonas aeruginosa lectins, binds to D-galactosamine, N-acetyl-D-galactosamine, alpha- and beta-galactosides as well as to D-galactose. Like these lectins it absorbs onto either untreated or enzyme (papain or neuraminidase) treated human red blood cells, but exhibits a considerable mitogenic activity towards human lymphocytes (predominantly T cells) only after their treatment with neuraminidase. This mitogenic stimulation of lymphocytes is inhibited by D-galactose and its derivatives. Despite the great similarity between them, the E. corallodendron, soybean, and Pseudomonas lectins differ in regard to the intensity of their agglutinating activity towards erythrocytes obtained from different animals and human donors of diverse ABO blood groups. This phenomenon may be attributed to the difference in the affinities of the three lectins to the various D-galactose derivatives and to their molecular properties.     

Purification and characterization of a galactan-reactive agglutinin from the clam Tridacna maxima (R?ding) and a study of its combining site.

1. A beta-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polylecyl larch galactan, D-galactosamine coupled to epoxy-activated Sepharose or acid-treated Sepharose. Elution with N-acetyl-D-galactosamine or lactose displaced the bound lectin, which appeared homogeneous by sedimentation analysis. On immunoelectrophoresis at pH8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the alpha-region. 2. On a alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Ioselectric focusing in solution revealed two peaks of pI4.05 and 4.25 and a shoulder, pI4.0, whereas at least three bands close together (pI3.9-4.3) were seen after electrofusing in gel. 3. The agglutinin is a glycoprotein with a mol.wt. of 470300 +/- 20000. Amino acid analysis revealed no methionine and a significant amount of half-cystine residues. 4. Tridacna lectin is a metalloprotein requiring Ca2+ for its haemagglutinating and precipitating activities. 5. In haemagglutination studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing beta-D-galactosyl residues reacted with the lectin to form precipitates both in gel and in solution. Inhibition experiments showed that N-acetyl-D-galactosamine was the best inhibitor of the agglutinin combining sites, followed by p-nitrophenyl beta-D-galactoside, methyl beta-D-galactoside, D-galactosamine and 60O-beta-D-galactopyranosyl-D-galactopyranose. On a molar basis, N-acetyl-D-galactosamine was 20-fold more active than D-galactose and nearly 10-fold more inhibitory than D-galactosamine. 7. Circular-dichroism studies showed that the lectin contains a relatively high proportion of beta-structure. 8. Mercaptoethanol treatment of the agglutinin followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed subunits with approx. mol.wts. of 10000, 20000 and 40000.     

A novel lectin from the sponge Haliclona cratera: isolation,characterization and biological activity

A lectin from the Adriatic sponge Haliclona cratera was purified by ion-exchange and gel chromatography. The molecular mass of the lectin is approximately 29 kDa. Purified lectin is rich in hydrophobic and basic amino acids and has an isoelectric point at pH 8.6. H. cratera lectin is relatively heat- and pH-stable. It agglutinates native and trypsinized, papainized and neuraminidase-treated human A, B, O, AB and sheep erythrocytes, and the hemagglutinating activity is independent of Ca(2+), Mn(2+) and Mg(2+) ions; D-galactose and N-acetyl-D-galactosamine are found to be moderate inhibitors of the activity. H. cratera lectin displays cytotoxic effect on HeLa and FemX cells and weak mitogenic effect on human T-lymphocytes pretreated with phytohemagglutinin (PHA).     

Isolation and partial characterization of an N-acetylgalactosamine-specific lectin from winter-aconite (Eranthis hyemalis) root tubers.

A lectin was isolated from root tubers of winter aconite (Eranthis hyemalis) by affinity chromatography on fetuin-agarose, and it was partially characterized with respect to its biochemical, physicochemical and carbohydrate-binding properties. The Eranthis hyemalis lectin is a dimeric protein (Mr 62000) composed of two different subunits of Mr 30000 and 32000, held together by disulphide bonds. It is especially rich in asparagine/aspartic acid, glutamine/glutamic acid and leucine, and contains 5% covalently bound carbohydrate. Hapten inhibition assays indicated that the winter-aconite lectin is specific for N-acetylgalactosamine. In addition, the lectin exhibits a pronounced specificity towards blood-group-O erythrocytes. The winter-aconite lectin is the first lectin to be isolated from a species belonging to the plant family Ranunculaceae. It appears to be different from all previously described plant lectins.     

A blood group A specific lectin from the seeds of Crotalaria striata

A lectin, monospecific for human blood group A red blood cells was extracted from seeds of Crotalaria striata and purified by molecular sieving on Sephadex G-100 and ion-exchange on DEAE-cellulose. A molecular mass of 30 kDa was determined by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions. Molecular sieving on a Superose 12 column indicated a molecular mass of 110 kDa, suggesting the tetrameric nature of the native protein. Amino-acid composition showed the presence of aminated carbohydrate residues on the lectin. N-terminal amino-acid sequencing showed a striking similarity with the N-terminal sequence of the lectin from Crotalaria juncea, which is blood-group non-specific. The potency order of agglutination inhibition with galactose containing monosaccharides was N-acetyl-D-galactosamine greater than D-galactose greater than D-galactosamine as found for blood-group-A-specific lectins from other species.     

The D-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates

A D-galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. D-Galactose, N-acetyl-D-galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80 degrees C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Purification of Phytohaemagglutinins from Phaseolus vulgaris and their Interaction with Plant Pathogenic Bacteria

Erythrocytes and different strains of plant pathogenic bacteria agglutinated with aqueous and saline 1M NaCl extracts from Phaseolus vulgaris L. seeds, which showed similar gel electrophoretic patterns of proteins. Haemagglutinating activity in aqueous extracts was inhibited by some carbohydrates, especially N-acetyl-D-galactosamine, D-galactose and lactose. The agglutinin in this extract was purified by utilizing its ability to bind to Pseudomonas pisi cells followed by its removal from the bacteria by 0.3M galactose. Mitogenic activity of purified agglutinin was calculated as incorporation of ¹²⁵I-iododesoxy-uridine into DNA of lymphocytes in vitro, which gave values between 25 and 50 μg/ml. The molecular weight of the subunits was found to be 31,500, estimated by mobility in SDS-PAGE. A solid phase competition-binding radioimmunoassay for bean agglutinin was developed in order to determine the affinity of this lectin in bean plants to phytopathogenic bacteria. The highest level of affinity was associated with the system formed by, Ps. pisi and lectin from fresh seeds.