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PubMed1979

Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.

Binding Sites↗

PubMed1996

Lectin histochemistry during in vitro capacitation and acrosome reaction in boar spermatozoa: new lectins for evaluating acrosomal status of boar spermatozoa.

Six peroxidase (HRP)-conjugated lectins have been used to analyze the distribution of carbohydrates in ejaculated boar spermatozoa during in vitro capacitation and acrosome reaction in air-dried preparations. The membranes of boar spermatozoa were positive to WGA and Con-A in the acrosome, middle piece and principal piece. PNA labelled the acrosomal region. DBA, UEA-I and LTA bound weakly or not to the spermatozoa. The lectin labelling pattern did not change during the in vitro capacitation. A diminution in the proportion of spermatozoa with acrosome positive to lectins PNA, WGA and Con-A was observed after coincubation with homologous immature oocytes. A positive correlation existed between the percentages of lectins bound to spermatozoa and the triple stain technique. The results show that peroxidase-conjugated lectins may be useful in determining acrosomal integrity of boar spermatozoa during in vitro capacitation and acrosome reaction.

Acrosome↗

PubMed2003

Purification of normal lymphocytes from leukemic T-cells by lectin-affinity adsorbents - correlation with lectin-cell binding.

Utilization of leukemic T-cells from normal ones, using lectin-affinity adsorbents, is described. CNBr-activated Sepharose 6MB was covalently coupled to Soybean (SBA) or Dolichos Biflorus Agglutinins (DBA), then serves as an affinity probe for separation of leukemic T-cells from normal lymphocytes. The normal lymphocytes were removed almost completely by phosphate buffered saline (Ca(2+) and Mg(2+) free) (PBS(-)) from lectin-affinity column. More than 80% of the leukemic T-cells were retained on the lectin-affinity adsorbent, whereas another 10-15% were easily removed by PBS(-). There was a very good linear correlation between percent of cells, retained on the lectin-affinity adsorbent and percent of cells, interacting with the respective free lectin (r=0.97 for SBA, and r=0.93 for DBA). The viability of normal lymphocytes was not influenced after passing through the columns. In the case of leukemic T-cells - about 90% of the easily removed cells were dead, and another 10% were viable cells, non-interacting with DBA or SBA.

Cell Separation↗

PubMed2003

Cloning and characterization of the Atlantic salmon serum lectin, a long-form C-type lectin expressed in kidney.

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.

Amino Acid Sequence↗

PubMedDate not supplied

Double-lectin site ricin B chain mutants expressed in insect cells have residual galactose binding: evidence for more than two lectin sites on the ricin toxin B chain.

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose binding is needed both for correlating structure-function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1 alpha or 2 gamma) and two double-site mutants (subdomains 1 alpha 2 gamma) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with alpha-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000-fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.

Binding Sites↗

PubMed2004

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PURPOSE: To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. METHODS: Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. RESULTS: The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. CONCLUSION: Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

Animals↗

PubMed1999

Purification and characterization of a cell-surface lectin (Lectin II) from Agrobacterium radiobacter NCIM 2443.

A lectin was isolated from Agrobacterium radiobacter cell surface and purified. It is a monomer of 40 kDa as shown by SDS-PAGE. The lectin has a pI of 9.15 and amino acid composition of the lectin shows that 44% of the amino acids are hydrophobic. The lectin agglutinates rabbit erythrocytes but does not agglutinate human erythrocytes. It does not show specificity for monosaccharides except for D-glucosamine. Fetuin and its N-linked glycopeptide also inhibit the activity of the lectin but greater inhibition is shown by locust bean gum and Nicotiana tobaccum (tobacco) tissue extracts.

Amino Acids↗

PubMed1996

Hapten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo.

The early chick blastoderm expresses two endogenous galactose-binding lectins of 14 kDa and 16 kDa. We have studied the effect the lectin hapten inhibitors thiodigalactoside and the synthetic neoglycoprotein lactosyl-bovine serum albumin as well as polyclonal anti-lectin antibodies on the development of early chick embryos cultured in a defined medium. Controls consisted of maltose, maltosyl bovine serum albumin and rabbit IgG. Embryos treated at the onset of cell migration during early gastrulation underwent blastoderm retraction with decrease in surface area. In addition, they exhibited a lack of demarcation between the presumptive embryonic area (area pellucida) and the presumptive extraembryonic area (area opaca). These blastoderms also lacked a primitive streak, that is, the structure that forms in the area pellucida during gastrulation as cell migrate to form the endodermal and mesodermal layers of the embryo. Embryos treated at later stages of gastrulation showed development similar to that of controls in that they were able to undergo early organogenesis. The results suggest that lectin mediated mechanisms are essential for the migratory movements of early gastrulation and that, at late gastrulation, other mechanisms exist in the embryo to compensate for lectin function.

Animals↗

PubMed2002

Production, properties and specificity of a new bacterial L-fucose- and D-arabinose-binding lectin of the plant aggressive pathogen Ralstonia solanacearum, and its comparison to related plant and microbial lectins.

The worldwide distributed plant aggressive pathogen Ralstonia solanacearum, which causes lethal wilt in many agricultural crops, produces a potent L-fucose-binding lectin (RSL) exhibiting sugar specificity similar to that of PA-IIL of the human aggressive opportunistic pathogen Pseudomonas aeruginosa. Both lectins show L-fucose > L-galactose > D-arabinose > D-mannose specificity, but the affinities of RSL to these sugars are substantially lower. Unlike Ulex europaeus anti-H lectin, but like PA-IIL and Aleuria aurantia lectin (AAL), RSL agglutinates H-positive human erythrocytes regardless of their type, O, A, B, or AB, and animal erythrocytes (papain-treated ones more strongly than untreated ones). It also interacts with H and Lewis chains in the saliva of "secretors" and "nonsecretors." RSL purification is easier than that of PA-IIL since R. solanacearum extracts do not contain a galactophilic PA-IL-like activity. Mass spectrometry and 35 N-terminal amino acid sequencing enabled identification of the RSL protein (subunit approximately 9.9 kDa, approximately 90 amino acids) in the complete genome sequence of this bacterium. Despite the greater phylogenetic proximity of R. solanacearum to P. aeruginosa, and the presence of a PA-IIL-like gene in its genome, the RSL structure is not related to that of PA-IIL, but to that of the fucose-binding lectin of the mushroom (fungus) Aleuria aurantia, which like the two bacteria is a soil inhabitant.

ABO Blood-Group System↗

PubMed1988

Lectin-carbohydrate interactions. Studies of the nature of hydrogen bonding between D-galactose and certain D-galactose-specific lectins, and between D-mannose and concanavalin A.

The binding of galactose-specific lectins from Erythrina indica (EIL), Erythrina arborescens (EAL), Ricinus communis (agglutinin; RCA-I), Abrus precatorius (agglutinin; APA), and Bandeiraea simplicifolia (lectin I; BSL-I) to fluoro-, deoxy-, and thiogalactoses were studied in order to determine the strength of hydrogen bonds between the hydroxyl groups of galactose and the binding sites of the proteins. The results have allowed insight into the nature of the donor/acceptor groups in the lectins that are involved in hydrogen bonding with the sugar. The data indicate that the C-2 hydroxyl group of galactose is involved in weak interactions as a hydrogen-bond acceptor with uncharged groups of EIL and EAL. With RCA-I, the C-2 hydroxyl group forms two weak hydrogen bonds in the capacity of a hydrogen-bond acceptor and a donor. On the other hand, there is a strong hydrogen bond between the C-2 hydroxyl group of galactose, which acts as a donor, and a charged group on BSL-I. The C-2 hydroxyl group of the sugar is also a hydrogen-bond donor to APA. The lectins are involved in strong hydrogen bonds through charged groups with the C-3 and C-4 hydroxyl groups of galactose, with the latter serving as hydrogen-bond donors. The C-6 hydroxyl group of the sugar is weakly hydrogen bonded with neutral groups of EIL, EAL, and APA. With BSL-I, however, a strong hydrogen bond is formed at this position with a charged group of the lectin. The C-6 hydroxyl groups is a hydrogen-bond acceptor for EIL and EAL, a hydrogen-bond donor for APA and BSL-I, and appears not to be involved in binding to RCA-I. The data with the thiosugars indicate the involvement of the C-1 hydroxyl group of galactose in binding to EIL, EAL, and BSL-I, but not to RCA-I and APA. We have also performed a similar analysis of the binding data of fluoro- and deoxysugars to concanavalin A [Poretz, R. D. and Goldstein, I. J. (1970) Biochemistry 9, 2890-2896]. This has allowed comparison of the donor/acceptor properties and free energies of hydrogen bonding of the hydroxyl groups of methyl alpha-D-mannopyranoside to concanavalin A with the results in the present study. On the basis of this analysis, new assignments are suggested for amino acid residues of concanavalin A [corrected] that may be involved in hydrogen bonding to the sugar.

Concanavalin A↗

PubMed1996

A multispecific saline-soluble lectin from the parasitic fungus Arthrobotrys oligospora. Similarities in the binding specificities compared with a lectin from the mushroom agaricus bisporus.

Several fungi can express high levels of saline-soluble and low-molecular-mass lectins that bind to glycoproteins such as fetuin and different mucins but not bind to any monosaccharides. In this paper, we report the binding specificities of such a lectin (designated AOL) isolated from the nematophagous fungus Arthrobotrys oligospora. The results show that AOL is a multispecific lectin that interacts with the following ligands: (a) Several sulfated glycoconjugates including sulfatide, dextran sulfate, and fucoidan. The specificity of this binding was indicated by experiments showing that none of the tested neutral- and sialic-acid-containing glycolipids, chondroitin sulfates B and C, heparin, and polyvinyl sulfate bound to AOL; (b) Phosphatidic acid and phospatidylglycerol, two out of several tested phospholipids. (c) N-linked and O-linked sugar chains bound to intact fetuin. The involvement of such sugar structures was demonstrated by analyzing the binding of AOL to chemically deglycosylated (trifluoromethanesulfonic acid) fetuin. Treating fetuin with O-glycosidase and N-glycosidase indicated that AOL bound to Gal beta GaLNAc alpha-Ser/Thr and to some N-linked complex sugars, respectively. Further assays demonstrated that AOL could interact with several other glycoproteins containing O-linked and/or N-linked sugar chains. The observations that AOL did not bind to free N-linked sugars isolated from fetuin, or to fetuin treated with trypsin or pronase, or to any of the tested neoglycoproteins and glycolipids with neutral- or sialic acid-containing sugars, indicated that the sugar chains need to be bound to an intact peptide backbone to interact with AOL. We have recently shown that the deduced primary structure of AOL has a high similarity to the sequence of a saline-soluble lectin isolated from the mushroom Agaricus bisporus (ABL) (Rosén, S., Kata, M., Persson, Y., Lipniunas, P. H., Wikström, M., van den Hondel, C. A. M. J. J., van den Brink, J. M., Rask, L., Hedén L.-O. and Tunlid, A., see companion paper). It is well known that ABL binds to Gal beta 3GaLNAc alpha-Ser/Thr, and in this paper we demonstrate that ABL binds to sulfatide, phosphatidic acid, phospatidylglycerol, and possibly also to the same N-linked complex sugars as AOL. The above data indicate that AOL and ABL are members of a novel family of fungal lectins sharing similar primary structure and binding properties.

Agaricus↗

PubMed1979

A statistical evaluation of the binding of ferritin-conjugated lectins to the surface of rat cells. Topographical variations as a function of lectin concentration and cell type.

Statistical procedures were utilized to evaluate the concentration dependence of labeling by ferritin-conjugated lectins on four different rat cells: hepatocytes, normal thymocytes, Friend virus-induced rat tumor cells and feline sarcoma virus-induced rat sarcoma cells. Labeling by ferritin conjugates of concanavalin A, wheat germ agglutinin and Ricinus communis agglutinins I and II was quantitated by counting the number of ferritin granules on 600 Angstrom membrane segments. Relationships between the arithmetic means and variances for sample populations from each cell and ferritin-lectin combination were used to define four types of topographical distributions: uniform/ordered, uniform/random, random and clustered. It was found that the distribution and/or density of surface-bound lectin was concentration-dependent for all four ferritin-lectins. The nature of this dependency was complex and varied with both lectin and cell type.

Animals↗

PubMed1983

Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes.

A new procedure is presented for the light microscopic demonstration of specific sugar sequences of oligosaccharides in glycoconjugates by lectins combined with the colloidal gold marker system. Tissue sections from aldehyde-fixed and paraffin embedded rat kidney were stained either in a one-step method with lectin directly bound to particles of colloidal gold or in a two-step method using non-labeled lectin and glycoprotein labeled with colloidal gold. In both methods the presence of lectin-binding sites in the tissue sections is revealed by the appearance of a red coloration that is due to the accumulation of gold particles. The high specificity of the technique is combined with a good sensitivity and resolution as demonstrated by a differential plasma membrane staining in renal epithelial cells. The lectin-gold or glycoprotein-gold complexes remain stable for months and produce a permanent nonbleaching staining.

Animals↗

PubMed1984

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Binding Sites↗

PubMed2001

A lectin from an ascomycete mushroom, Melastiza chateri: no synthesis of the lectin in mycelial isolate.

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.

Amino Acid Sequence↗

PubMed2002

Distribution and molecular evolution of rhamnose-binding lectins in Salmonidae: isolation and characterization of two lectins from white-spotted Charr (Salvelinus leucomaenis) eggs.

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.

Amino Acid Sequence↗

PubMed1985

Comparative studies of normal and genetically hyperplastic lens epithelia II: lectin-induced cell agglutination and 125I-lectin uptake by cells.

The reactions of lectins with dissociated and cultured epithelial cells from hyperplastic lenses of two unrelated chick strains, Hy-1 and Hy-2, characterised by hyperplasia of the lens epithelium, and lenses of a normal genotype (N) were investigated using four different lectins. Three methods of monitoring lectin-binding to cell surfaces were employed. Each of the four lectins used showed an individual pattern of reactivity to separated membrane components. Data obtained with the three labelling methods showed the same trend viz: increased agglutinability and high affinity for binding of all four lectins by Hy-1 and Hy-2 cells. These results suggest that Hy-1 and Hy-2 lens epithelial cells are characterised by alteration in their cell surfaces.

Agglutination↗

PubMed1985

Comparison of binding proteins on the surface of murine tumor cells for two lectins active in the lectin-dependent macrophage-mediated cytotoxic reaction.

The binding proteins for Sarcophaga lectin and wheat germ agglutinin on the surface of Ehrlich ascites tumor cells were compared. Studies with antibody against the binding protein for Sarcophaga lectin showed that these two binding proteins are different. Since these two lectins are both active in the lectin-dependent macrophage-mediated cytotoxic reaction with Ehrlich ascites tumor cells as target cells, there must be multiple proteins on the surface of target cells that can trigger cytolytic reaction in response to different lectins in the presence of macrophages.

Animals↗