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.     

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.     

Lectins as tools in glycoconjugate research

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate–protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin–carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin–glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, β-elimination), which extend the applicability of these methods, are also described.     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Purification and characterization of mannose/glucose-specific lectin from seeds of <Emphasis Type="Italic">Trigonella foenumgraecum</Emphasis>

A lectin present in seeds of Trigonella foenumgraecum was isolated and purified by acid precipitation, salt fractionation, and affinity chromatography on mannan cross-linked agarose. SDS-PAGE revealed a single band corresponding to a molecular weight of 27,350 daltons. The lectin agglutinated trypsin-treated rat erythrocytes. Sugar specificity as determined by hemagglutination inhibition assay indicated that the lectin belongs to a glucose/mannose-specific group. The reaction of the lectin with glycoprotein was affected by pH changes. The carbohydrate binding specificity of the lectin was investigated by turbidity and activity measurements. As the lectin belongs to the Leguminoceae family, the specificity of the lectin for glucose/mannose renders it a valuable tool for Rhizobium-legume symbiosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 52–57.     

Characterization of an endogenous quail intestinal lectin

Soluble extracts of quail intestine scrapings contain a lectin activity specific for chicken and rabbit trypsinized, glutaraldehyde-fixed erythrocytes. The lectin displayed a specificity for the simple sugar haptens lactose and galactose and for mucin. Quail lectin was purified by affinity chromatography on either asialofetuin- or mucin-Sepharose, followed by DEAE-Sepharose chromatography, and demonstrated an apparent molecular weight of 14,500 on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a pI of 6.2 upon isoelectric focusing. Immunohistochemical localization of this lectin in the intestine was carried out using polyclonal antibody raised in rabbits and tested for specificity in Western blots. Immunoperoxidase staining for quail lectin showed the lectin to be prominent in secretions at the mucosal surface and in goblet cells.     

Analysis of the carbohydrate binding specificity of the mushroom Pleurotus ostreatus lectin by surface plasmon resonance

The sugar binding specificity of the mushroom Pleurotus ostreatus lectin (POL) was analyzed by surface plasmon resonance. The lectin was immobilized to a sensor chip, and asialo-bovine submaxillary mucin (asialo-BSM), one of the most potent inhibitors in the hemagglutination inhibition assay, tightly bound to the lectin. The binding specificity of various mono- or oligosaccharides to the lectin was evaluated by the coinjection method. The dissociation of asialo-BSM was promoted by injection of some haptenic saccharides. For the most part, the order of acceleration ability of the sugars to the dissociation in the coinjection experiment agreed with that of the inhibitory potency of each sugar evaluated by the hemagglutination inhibition assay. In conclusion, POL recognized a galactosyl residue, and the specificity was increased by substitution at the C-2 position of the galactosyl residue with a fucosyl or acetylamino group. This method using the coinjection method proved useful in analysis of carbohydrate-lectin binding specificity.     

7-Deoxyloganin 7-hydroxylase in Lonicera japonica cell cultures

A lectin was isolated from the mushroom Mycoleptodonoides aitchisonii by means of affinity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel filtration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of four identical 16 kDa subunits and the molecular mass of the intact lectin was estimated to be 64 kDa by gel filtration. In a hemagglutination inhibition assay, it exhibited strong sugar-binding specificity towards asialo-BSM among the mono- or oligo-saccharides and glycoproteins tested. The binding specificity of the lectin was also examined by surface plasmon resonance analysis.     

A new type of cereal lectin from leaves of couch grass (Agropyrum repens)

Extracts from couch grass (Agropyrum repens) leaves contain relatively high lectin concentrations. Preliminary experiments with crude extracts indicated that the leaf lectin differs from the embryo lectin of the same species and other Gramineae embryo lectins with respect to its sugar and blood group specificity, and serological properties. A comparison of the biochemical, physicochemical and biological properties of purified lectins from couch grass leaves and embryos, and wheat germ agglutinin revealed that the leaf lectin has the same molecular structure as the embryo lectins. It is a dimer composed of two identical subunits, which, however, are slightly larger than embryo lectin subunits. Structural differences between both couch grass lectins were further inferred from in vitro subunit exchange experiments and serological analyses. Whereas the embryo lectin readily forms heterodimers with embryo lectins from other cereal species and also is serologically indistinguishable from them, the leaf lectin does not exchange subunits with the same embryo lectins and is serologically different. In addition, couch grass leaf lectin exhibits specificity for N-acetylgalactosamine and agglutinates preferentially blood-group-A erythrocytes whereas the embryo lectin is not inhibited by N-acetylgalactosamine and exhibits no blood-group specificity. It was observed also that the lectin content of couch grass leaves varies enormously during the seasons.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

The lectin from leaves of Japanese cycad, Cycas revoluta Thunb. (gymnosperm) is a member of the jacalin-related family.

A novel lectin was isolated from leaves of the Japanese cycad, Cycas revoluta Thunb. (gymnosperm), and its characteristics including amino acid composition, molecular mass, carbohydrate binding specificity and partial amino acid sequences were examined. The inhibition analysis of hemagglutinating activity with various sugars showed that the lectin has a carbohydrate-binding specificity similar to those of mannose recognizing, jacalin-related lectins. Partial amino acid sequences of the lysylendopeptic peptides shows that the lectin might have a repeating structure and belong to the jacalin-related lectin family.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Trypanosoma brucei gambiense and T. b. rhodesiense: concanavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.     

Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 μg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.     

Subunit structure of Vicia graminea anti-(blood-group N) lectin.

The subunit of the Vicia graminea lectin with blood-group-N specificity was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in 6M-guanidinium chloride, and its molecular weights was found to be 25 000. The unique N-terminal sequence fof the first nine residues of the lectin confirmed that Vicia lectin consists of four identical chains non-covalently linked. Finally the microheterogeneity of the lectin shown by analytical isoelectric focusing is discussed.     

Lectin extracts of champedak seeds demonstrate selective stimulation of T lymphocyte proliferation.

The effect of extracts of champedak (Artocarpus integer) seed lectin on the proliferation of normal human lymphocyte was investigated. The IgA1 binding lectin was demonstrated to stimulate the proliferation of human peripheral blood mononuclear cells. Action of the lectin on enriched T and B cell populations demonstrated T lymphocyte specificity. The lectin was not mitogenic to B lymphocytes. Optimal stimulation of proliferative response was achieved when cells were subjected to 5 days exposure to the crude lectin at 20 micrograms/ml.     

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.     

Trypanosoma brucei gambiense and T. b. rhodesiense: Concanavalin A Binding to the Membrane and Flagellar Pocket of Bloodstream and Procyclic Forms1

ABSTRACT We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3–16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed >95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.