Canavalia Ensiformis Lectin Research Articles

White blood cells of peripheral blood with ConA-positive glycotopes in patients with chronic leukemia

141 Tumor growth progression of blood cells occurs due to changes in their genetic apparatus, which affects not only the cells morphological characteristics, but also their functional activity which to a greater extent depends on the membrane surface structures, a significant part of which is of glycoprotein nature. Complex type N-glycans are components of surface glycoproteins in the most of leukocytes. Thus, the study of changes in carbohydrate determinants of glycoproteins on the surface of leucocytes in tumorigenesis can help to reveal the mechanisms of this process. The aim of our study was to investigate the monocytes and granulocytes cytoplasmic membrane N-glycosylation in patients with chronic leukemia. The object of the study were blood cells of patients with chronic lymphocytic leukemia (n = 12) and polycythemia vera (n = 15) aged 58–66 years. Healthy hematologic volunteers (n = 15) aged 55 to 65 years were in the control group. N-glycan exposure on monocytes and granulocytes was investigated by flow cytometer Beckman Сoulter EPICS with Canavalia ensiformis lectin – Con A conjugated with fluorescent labels. The number of dead cells was monitored by means of binding them with propidium iodide. The result has been analyzed with FC Express. According to our data, levels of ConA-positive monocytes and granulocytes were 9,9 ± 1,0% and 32,7 ± 3,2%, respectively, in peripheral blood of healthy persons. The level of ConA-positive monocytes decreased to 31,0 ± 2,3% and the number of ConA-binding granulocytes increased to 66,7 ± 3,8% in patients with chronic lymphocytic leukemia compared with the norm. The number of ConA-positive monocytes decreased 3.3 times, and the level of granulocytes interacting with Canavalia ensiformis lectin slightly increased relative to control in polycythemia vera patients. There is significant increase in Con A-positive epitopes on granulocytes in patients with chronic lymphocytic leukemia and polycythemia vera compared with the control group, and absence of any difference in Con A-positive epitopes on monocytes. 100 times increase of the number of ConA-binding glycotopes is observed on the surface of granulocytes in peripheral blood of patients with polycythemia vera, and 3,3 times increase in patients with chronic lymphocytic leukemia, which may serve as an additional marker for the diagnosing these diseases.

Mechanism of entomotoxicity of the Concanavalin A in Rhopalosiphum padi (Hemiptera: Aphididae).

The toxicity effect of Concanavalin A ( Canavalia ensiformis lectin, ConA) to bird cherry-oat aphid, Rhopalosiphum padi L. (Hemiptera: Aphididae), was investigated in the laboratory by using artificial diets containing ConA concentrations. Bird cherry-oat aphid performance was affected by the presence of Con A in artificial diets. The lectin added into the liquid diet increased the prereproductive period, mortality, and the average time of generation development ( T ) and decreased fecundity and the intrinsic rate of natural increase ( rm ). In attempt to unravel the mode of action of ConA, the interaction of the lectin with insect gut and the effect of ConA on feeding behavior were investigated. Extract of gut of treated grain aphid demonstrated DNA fragmentation, and this was accompanied with an increase in caspase 3 activity. Moreover, addition of ConA to the sucrose–agarose gels reduced salivation and passive ingestion of fluids from the gel. The results indicate that the insecticidal activity of ConA on R. padi may involve effects on death of the gut epithelial cells and effects on feeding behavior. This can be employed to create plants that are resistant to aphids.

Rachycentron canadum (cobia) Lectin Promoted Mitogenic Response in Mice BALB/c Splenocytes

The mitogenic lectins are invaluable tools to study the biochemical changes associated with lymphocyte activation and proliferation of various immune cells. Rachycentron canadum lectin (RcaL) was detected and purified from serum of cobia fish. The aim of this study was to evaluate the proliferative response and cytokine production in splenocytes of mice in vitro stimulated with RcaL lectin; Canavalia ensiformis lectin (Con A) was used as positive control. A high proliferation index was induced by RcaL in relation to control cells. Furthermore, RcaL induced higher IL-2 and IL-6 production in relation to control. The cell viability was 90% in splenocytes treated with RcaL lectin, but RcaL promoted significant late apoptosis after 24 and 48 h in relation to control. RcaL induced proliferative responses suggesting that this lectin can be used as a mitogenic agent in immunostimulatory assays.

Characterization of the lectin binding pattern in human spermatozoa after swim-up selection.

Capacitation is characterized by a hyperactivated pattern of sperm motility. The acquisition of highly motility is present in the early stages of capacitation. Sperm progressive motility is one of the most important parameters for determining the suitability of semen for processing. However, previous studies have shown that some sperm showing good motility have membrane damage. The aim of our study was to characterize the lectin staining pattern on the sperm plasma membrane of unselected and selected human sperm of normozoospermic donors. Sperm selection was performed by the swim-up technique. Fourteen samples from healthy consenting donors classified as normozoospermic according to the World Health Organization were used. We observed changes in the distribution of the carbohydrate residues after the swim-up selection. With Triticum vulgaris, the most abundant pattern was dotted labeling all over the head plasma membrane in the unselected sperm. However, this lectin was distributed homogenously over the acrosomal region after selection. With Arachis hypogaea, the most abundant pattern in fresh sperm was a highly stained acrosomal region. In the highly motility sperm population, the most frequent pattern was dotted fluorescence on the acrosomal region and a highly stained equatorial segment. Meanwhile, with the Aleuria aurantia and Canavalia ensiformis lectins, the most representative patterns were the same before and after the swim-up selection. Our data indicate that modifications which occur in carbohydrate residues during swim-up selection could be important for the regulation of progressive motility and prepare the sperm for capacitation.

N-Glycosylation Pattern of Human Placental Insulin-Like Growth Factor and Insulin Receptors in Well-Controlled Pregestational Diabetes Mellitus

N-Glycosylation Pattern of Human Placental Insulin-Like Growth Factor and Insulin Receptors in Well-Controlled Pregestational Diabetes MellitusDiabetes mellitus is a complex disease that leads to alterations in the glycosylation of proteins. Insulin-like growth factor and insulin receptors are involved in the regulation of fetal and placental growth and development. In this work the N-glycans of these receptors, originating from placentas obtained from pregnancies complicated by pregestational insulin dependent diabetes mellitus, were studied. Diabetic mothers were under regular insulin therapy. Solubilised membrane samples from healthy and diabetic placentas were analysed using lectin-affinity chromatography. N-glycans bound to insulin-like growth factor and insulin receptors were studied in terms of their interaction with eleven agarose-immobilised lectins: wheat germ agglutinin, succinylated wheat germ agglutinin,Ricinus communisagglutinin I,Sambucus nigraagglutinin,Erythrina cristagallilectin,Ulex europaeusagglutinin,Lens culinarisagglutinin,Canavalia ensiformislectin,Phaseolus vulgariserythro- and leukoagglutinin andMaackia amurensisagglutinin. A very similar type of N-glycans and content of the terminal saccharide residues were found in both groups of placentas. The results of this work suggest that the tight glycemic control may prevent alterations in the glycosylation of insulin-like growth factor and insulin receptors, thus maintaining physiological homeostasis during pregnancy and fetal growth.

Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: New insights into the understanding of the structure–biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Leguminosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/CGL:Man(α1-2)Man(α1-O)Me, ConM/CGL:Man(α1-3)Man(α1-O)Me and ConM/CGL:Man(α1-4)Man(α1-O)Me, which crystallized in different conditions and space group from the native proteins.The structures were solved by molecular replacement, presenting satisfactory values for Rfactor and Rfree. Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe.

Lectinocytochemical detection of apoptotic murine leukemia L1210 cells.

Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about its changes during apoptosis. We investigated whether specific changes in the expression of plasma membrane glycoproteins take place during apoptosis and whether these changes could be used for a quantitative estimation of apoptosis. Lectin cytochemical study of normal and apoptotic murine leukemia cells of the L1210 line was done. Horseradish peroxidase-labeled Laburnum anagyroides bark agglutinin, Phaseolus vulgaris agglutinin, Pisum sativum lectin (PSL), Ricinus communis agglutinin (RCA-120; 120 kDa), Solanum tuberosum agglutinin, Triticum vulgaris lectin (wheat germ agglutinin), Viscum album agglutinin, Canavalia ensiformis lectin (concanavalin A), and Helix pomatia lectin were used to compare specific glycoprotein expressions in normal and apoptotic murine leukemia cells of the L1210 line sensitive (L1210) and resistant (L1210R) to apoptosis induction by cisplatin. The data demonstrated significantly increased binding of alpha-D-mannose-specific PSL lectin (P<0.01) and beta-D-galactose-specific RCA-120 lectin (P<0.001) by the apoptotic cells of the L1210 and L1210R lines in comparison with the intact cells. That binding was shown to be specific because it was blocked by the corresponding inhibitory sugars. Lectins specific to alpha-D-mannose (PSL) and beta-D-galactose (RCA-120) can be used to distinguish between native and apoptotic murine leukemia L1210 cells and to quantitatively estimate apoptosis in a population of these cells.

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1+/SP-A+ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.

AFFINITY CONCENTRATION OF LISTERIA MONOCYTOGENES CELLS ON CONCANAVALIN A‐COATED POLYESTER CLOTH AND SUBSEQUENT DETECTION BY THE POLYMERASE CHAIN REACTION

The Canavalia ensiformis lectin concanavalin A (con A) was immobilized on macroporous polyester cloth to form an inexpensive high surface area adsorbent (con A‐cloth) for the affinity concentration of bacterial cells in aqueous suspensions. When the con A‐cloth was packed into a 1 ml pipette tip, the resulting mini‐column could be used to concentrate large volumes of dilute L. monocytogenes cell suspensions, followed by the polymerase chain reaction (PCR) amplification of L. monocytogenes‐specific hly A sequences from lysates of the captured cells. This improved the effective sensitivity of the PCR as compared to the assay of L. monocytogenes in unconcentrated suspensions. Several enrichment broths (Fraser Broth, Listeria Enrichment Broth and Brain Heart Infusion Broth) were found to be inhibitory to the PCR of L. monocytogenes when introduced directly into the reaction mixture. This inhibitory effect was completely eliminated when the L. monocytogenes cells were captured on the con A‐cloth and washed to remove the enrichment broth components prior to performing the PCR. Since the lectin con A is reactive with a broad variety of Gram positive and Gram negative bacteria, this simple and inexpensive affinity concentration method should be applicable to the PCR detection of other pathogens in enrichment cultures.

The major Kurloff cell glycoproteins: lectin affinities, glycosidase susceptibilities and relationship with the sialylated acid phosphatases of the Kurloff body

Urea-soluble fractions from purified Kurloff cells (KC) were analysed by affinoblotting. Lectin reactivities were quasi-exclusively confined to the 30–35 kDa major glycoproteins (mGPs) (responsible for the PAS positivity of the Kurloff body) with strong affinities for Canavalia ensiformis lectin, Phaseolus vulgaris erythroagglutinin and Sambucus nigra (SNA), Pisum sativum, Triticum vulgaris and Ulex europeus agglutinins. These data were consistent with the presence, among the KC mGPs, of large amounts of complex or hybrid N-glycosylproteins, in particular with Neu5Acα2,6Gal/GalNAc sequences, fucosyl residues and bisected residues. Their oligosaccharide sequences belong to more than one class, since some of these lectin reactivities had to be borne by distinct N-linked oligosaccharide chains. Before further analysis, KC mGPs were separated from other highly anionic glycoconjugates, by DEAE-cellulose chromatography. Their abundant potential RCA-binding sites masked by sialic acid were then revealed after neuraminidase (sialidase) or dilute acid pre-treatment. In remaining consistent with their lectin affinities, some KC mGPs were found to be PNGase F sensitive, while, either desialylated or not, they were all O-glycanase insensitive. Finally, by compound zymography and affinoblotting, the SNA-reactive fraction of KC mGPs was shown to correspond to denatured forms fo the two zymographic size populations (190 kDa and 500 kDa) of KC acid phosphatases.

Lectin binding sites on the surfaces of the pneumonocytes in human neonatal lung.

The surface coating of the pneumonocytes in human neonatal lung was studied by means of an electron microscope technique. Slices of aldehyde-fixed lung tissue were labelled with a horseradish peroxidase conjugate of one of the following lectins: Dolichos biflorus lectin, Triticum vulgaris lectin, Canavalia ensiformis lectin (concanavalin A), Limulus polyphemus lectin, Lotus tetragonolobus lectin and Arachis hypogaea lectin. The tissue slices were then incubated in a diaminobenzidine-hydrogen peroxide medium and then postfixed in an osmium tetroxide solution. It was found that the type I and type II pneumonocytes were strongly labelled with the lectins of Triticum vulgaris, Canavalia ensiformis, Limulus polyphemus and Arachis hypogaea. The type I pneumonocytes were also strongly labelled with Dolichos biflorus lectin but the staining of type II cells was relatively weak with this agent. Neither type of epithelial cell was labelled with Lotus tetragonolobus lectin conjugate. These results suggest that the surface coating of the pneumonocytes in human neonatal lung contains the following carbohydrate groups: N-acetylgalactosamine, N-acetylglucosamine, alpha-D-mannose, beta-D-galactose and sialic acid.

The structure of the lentil (Lens culinaris) lectin. Amino acid sequence determination and prediction of the secondary structure.

The subunit structure and complete amino acid sequence of the lectin extracted from Lens culinaris (LcL) seeds was determined. In previous studies, the primary structure of the alpha-chain (Mr = 5,710) was shown to be homologous to the alpha-chain of the lectin from Pisum sativum, the Vicia cracca glucose-specific lectin, and a region in the middle of the concanavalin A sequence (residues 70-121). The complete amino acid sequence of the beta-chain (Mr = 17,572) has been determined from 11 tryptic peptides, 4 peptides derived by chemical cleavage of the beta-chain at its three tryptophan residues, 11 peptides obtained after digestion with Staphylococcus aureus protease, and 5 tryptic peptides from the succinylated polypeptide chain. The extensive homologies by alignment of the alpha- and beta-chains of the L. culinaris lectin with portions of concanavalin A situated between 1 to 45 and 70 to 237, suggest that the L. culinaris and Canavalia ensiformis lectins have evolved from each other. A comparison was made between the secondary structure of the C. ensiformis lectin and the probable secondary structure of the L. culinaris lectin as predicted by two different methods. The results indicate that the folding of these two polypeptides has been particularly well conserved during evolution. It is suggested that the L. culinaris lectin is synthesized as a single polypeptide chain and cleaved subsequently into two or possibly three fragments, two of which would be alpha and beta and the third a fragment homologous to portion 46 to 69 in concanavalin A. As in favin, the amino acids postulated to be involved in the formation of the hydrophobic cavity and the sugar and metal binding sites are highly conserved in L. culinaris lectin.