Cratylia Floribunda Research Articles

The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.

Influence of fetal bovine serum on cytotoxic and genotoxic effects of lectins in MCF‐7 cells

Canavalia ensiformis (ConA), Canavalia brasiliensis (Conbr), and Cratylia floribunda (CFL) lectins have exhibited glucose-mannose binding specificity. We investigated the effect of fetal bovine serum (FBS) concentrations (1, 5, 10, and 20%) on the cytotoxic effect of these lectins against breast tumor cell line MCF-7. Cell viability was examined using the MTT reduction assay. When cells were grown in a medium supplemented with a higher serum concentration (10 or 20%), all lectins were much less toxic. When we used 1% FBS, it was possible to achieve a concentration-dependent activity by all examined lectins, with an IC(50) of 3.5, 25, and 60 µg/mL for ConA, Conbr, and CFL, respectively. All lectins incubated with 1% FBS induced apoptosis and DNA damage in MCF-7 cells. We conclude that ConA, Conbr, and CFL lectins' cytotoxic and genotoxic effects were observed only at low concentrations of serum.

Toxicity of some glucose/mannose-binding lectins to Biomphalaria glabrata and Artemia salina

Schistosomiasis or bilharzia, which affects millions of people living in Africa, Asia and Latin America, is closely associated with certain species of aquatic snails. One way of attacking the disease is to eradicate the host snails. Molluscicidal activities of natural compounds are especially important in the widespread control of this tropical disease. As part of our search for natural compounds with molluscicidal properties for the vector control of schistosomiasis, we are now evaluating for the first time the toxicity of the plant lectins from Canavalia brasiliensis (ConBr), Cratylia floribunda (CFL), Dioclea guianensis (Dgui), Dioclea grandiflora (DGL) and Dioclea virgata (Dvir) to Biomphalaria glabrata Say and Artemia salina Leach. Results indicate that all the samples were toxic to A. salina Leach, some of them with values of lethal concentration that kills 90% of the population (LC 90) < 10 μg mL −1. They are also active against B. glabrata Say, killing 100% of adult snails, at a concentration of 50 μg mL −1. The lectins CFL and Dgui possess properties lethal to mollusks, with values of LC 90 = 50.3 μg mL −1 and LC 90 = 41.0 μg mL −1, respectively.

Crystal structures of Cratylia floribunda seed lectin at acidic and basic pHs. Insights into the structural basis of the pH-dependent dimer–tetramer transition

Structural determinants underlaying the pH-dependent dimer–tetramer transition of Diocleinae lectins were investigated from the structures of Cratylia floribunda seed lectin crystallized in conditions where it exist as a dimer (pH 4.6) or as a tetramer (pH 8.5). The acidic (aCFL) and the basic (bCFL) tetramers superimpose with overall r.m.s.d. of 0.53 Å, though interdimer contacts are drastically reduced in aCFL, and the r.m.s.d. for the superposition of the 117–120 loops of aCFL vs. the bCFL tetramer is 1.29 Å. Our data support the view that His51 plays a role in determining the conformation of the central cavity loops and that interdimer contacts involving ordered loop residues stabilize the canonical, pH-dependent tetramer. In the bCFL tetramer, hydrogen bonds between Asn118 and Thr120 of monomers A and D and residues Ser66, Ser108, Ser110, and Thr49 of the opposite monomer stabilize the canonical, pH-dependent tetrameric lectin structure. In CFL, Asn131 makes intradimer contacts with Asn122 and Ala123. In comparison, His131 in Dioclea grandiflora lectin establishes a network of interdimer interactions bridging the four central loops of the pH-independent tetramer. Our data provide new insights into the participation of specific amino acid residues in the mechanism of the quaternary association of Diocleinae lectins.

In vitro inhibition of Streptococci binding to enamel acquired pellicle by Plant Lectins

Initial colonization of the tooth surface by streptococci involves the attachment of these bacteria to adsorbed salivary components of the acquired pellicle. In dental biofilm this adhesion may also involve lectin-like components, present on the surface of the organisms, which bind to complementary carbohydrates on the surface of the tooth. Therefore, this work aimed to evaluate the potential of six lectins, extracted from seeds of Leguminosae family members, to inhibit the adherence of five streptococci species to acquired pellicle in vitro. The lectins used in this work were extracted from Canavalia ensiformis, Canavalia brasiliensis, Dioclea violacea, Dioclea grandiflora, Cratylia floribunda and Vatairea macrocarpa. Fluorescence micrography was employed to visualize the ability of FITC-labeled lectins to attach to acquire pellicle. Adherence inhibition was performed on saliva-coated microtiter plates at which lectins solutions were previously incubated followed by incubation with the oral streptococci. Glucose-mannose specific lectins attached to acquired pellicle with high intensity, while galactose specific lectins, from V. macrocarpa, exhibits low intensity attachment. All lectins were able to inhibit the adherence of the microorganisms tested (p < 0.01). Our results suggest that lectins may be useful in anti adhesion therapeutics.

Kinetic sedimentation of Rhizobium-aggregates produced by leguminous lectins

Lectins from Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Vatairea macrocarpa (VML) and Phaseolus vulgaris (PHA) aggregate Rhizobiumbacteria. The relationship between specific sedimentation rate, ν′ (based on bacterial dry biomass) of bacterial aggregates and lectin concentrations was hyperbolic and showed bacterial surface affinity by lectins. R. tropici (Rt), R. leguminosarum bv. phaseoli (Rlp) and R. etli (Re) surfaces showed predominantly receptors of galactosidic nature. The Rt surfaces showed very high affinities (k s = ±8.6 × 10−8 ag lectin protein ml−1) by Gal-specific lectins (PHA and VML), and very low affinities (ks=± 4.9 × 10−6) by Glc-specific lectins (CnBr and CFL). The Rlp surface had intermediate affinities by lectins. The Re surface showed high affinities by PHA (ks= ±1.26 × 10−8) and intermediate affinities by VML, CnBr and CFL. The relationship between sedimentation specific ν′′ (based on lectin weight) and bacterial density was a sigmoid and showed lectin affinity by Rt surfaces. The bacterial sedimentation showed positive cooperative binding of lectins. The V′′max induced by Glc-specific lectins was ±20 of that produced by Gal-specific lectins. The PHA affinity (ks= 1.19 mg dry biomass ml−1) was larger than VML (ks = 1.23). The Glc-specific lectin affinities were smaller than those of Gal-specific. The apparent binding site number of lectins (napp) was: 2.7-PHA; 2.2-VML; 3.2-CFL and 3.2-CnBr. The dissociation constant, ks, of lectin-binding kinetics decreased with sugar-hapten treatment (10 μM). The napp decreased in PHA and CFL, increasing in VML + sugar-hapten treatment. This study showed that there is a difference in Rhizobium surfaces for lectin binding.

Respiratory stimulus in Rhizobium sp. by legume lectins

High molecular weight lectins (> 100 kDa) from seeds of the legumes Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Phaseolus vulgaris (PHA) and Vatairea macrocarpa (VML), temporarily stimulate the respiration of Rhizobium tropici-CIAT899 and R. etli-CFN42. These stimulants were significant (P 2.85 mg dry biomass ml−1), having at least 6200 molecules of lectins per bacteria. The VML (20 μg ml−1), induced specific O2 demand of 2.3–2.5 ηM O2 min−1 mg dry biomass−1, in CFN42 and CIAT899, respectively. However, CnBr, CFL and PHA induced smaller demands of O2 (5×), in both strains. The order of affinities of the lectins was approximately VML > PHA > CFL > CnBr, with regard to respiratory stimuli in CIAT899 strain. The co-administration of 10 μg VML ml−1 and 9.8 ηM galactose, in CIAT899 suspensions, reduced the respiratory stimuli significantly in relation to the treatment with VML alone. These respiratory stimuli, induced by the lectins, increase the significance of the interaction lectin × Rhizobium in terms of bacterial physiology. Its understanding could be important in relation to bacterial symbiotic behaviour.

Evidence of an endogenous lectin receptor in seeds of the legume Cratylia floribunda

Cratylia floribunda seeds were ground and the clean crude saline extract was fractionated into albumin, globulin, prolamin, acidic and basic glutelin protein fractions. These protein fractions were examined for the presence of an endogenous lectin receptor by SDS-polyacrylamide gel electrophoresis, western blot, affinity chromatography on a Sepharose 4B-Cratylia floribunda (CFL) lectin column and kinetic analysis in real time by surface plasmon resonance (SPR). Prolamin was the richest protein fraction although very poor in haemagglutinating activity. Basic glutelin was far the less interesting fraction for lectin activity and protein content, even though this fraction contains considerable amounts of carbohydrates. Lectin was present in all protein fractions as estimated by haemagglutinating assays but basic glutelins were almost devoid of lectin activity. Except for prolamins, protein bands were detected by SDS-PAGE in all other fractions. Western blot using digoxigenin labelled Con A revealed a single band in the albumin, globulin, acidic and basic glutelin fractions, which specifically interacted with ConA. This band migrated exactly at the same position in such fractions and seemed to be more important in the globulins. Affinity chromatography of the protein fractions on a Sepharose-CFL column yielded a peak, which was only recovered after elution with acidic buffered solution or with an alpha-D-mannose solution and the monosaccharide was recognized by the lectin. These results were fully corroborated by real time interaction of immobilized CFL with the different soluble protein fractions suggesting the presence of a lectin receptor within albumins, globulins and basic glutelins. As a whole, the results suggest that the lectin from Cratylia floribunda recognizes a soluble endogenous glycosylated receptor through an interaction mediated by its carbohydrate-binding site.

Thermodynamic Binding Studies of Lectins from the Diocleinae Subtribe to Deoxy Analogs of the Core Trimannoside of Asparagine-linked Oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy.

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.

EVALUATION OF THE PROTEOLYTIC SUSCEPTIBILITY OF THREE LECTINS FROM SUBTRIBE DIOCLEINAE USING ENZYMATIC ACTION, HEAT TREATMENT AND MOLECULAR MODELING

The enzymatic digestion of the lectins from Canavalia brasiliensis, Dioclea grandiflora and Cratylia floribunda seeds was investigated by heat treatment and gel filtration chromatography. The potential sites of lectin cleavage by pepsin and trypsin was then determined by molecular modelling of the three-dimensional structure of D.grandiflora lectin. The lectins were shown to be susceptible to proteolytic cleavage using peptide mapping with the FPLC system. However, new peptides arose when hydrolysis was followed by heat treatment at 73C. According to the molecular model of D.grandiflora lectin, there are numerous sites for pepsin and trypsin proteolysis on the surface of the protein. It is suggested that when subjected to enzymatic action, the resulting peptides interact in order to retain the general folding of the protein. This is attributed, at least in part, to the numerous hydrogen bonds and hydrophobic interactions occurring within the inner protein structure. After heat treatment the additional cleavage sites become available and the lectins become completely inactivated.

Molecular characterization and crystallization of Diocleinae lectins

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.

Primary Structure and Kinetic Interaction with Glycoproteins of The Lectin From Seeds of Cratylia Flor/Bunda

Abstract: The complete 236-amino-acid sequence of the glucose/mannose specific lectin from seeds of Cratylia floribunda (CFL) was determined by automated Edman sequencing of overlapping proteolytic peptides purified by HPLC after digestion of the lectin with endoproteinases Lys-C, Asp-N, trypsin and chymotrypsin. Mass spectrometry confirmed the sequence analysis and showed that CFL consists of a mixture of full length, single­ chain polypeptide (a-chain.-25397 ± 3 Da) and its• noncovalently associated p (residues 1-11 , 12847 ± 2 Da) and y (residues 119-236, 12568 ± 1 Da) fragments. The primary structure of Cratylia floribunda lectin has extensive amino acid sequence homology with those of lectins from species of the taxonomically related genera Canavalia and Dioclea. However, using surface plasmon resonance, CFL and ConA, the seed lectin from Canavalia ensiformis, displayed distinct kinetic interactions with glycoproteins, indicating structural differences in their extended glycan binding-sites.

Diocleinae Lectins Are a Group of Proteins with Conserved Binding Sites for the Core Trimannoside of Asparagine-linked Oligosaccharides and Differential Specificities for Complex Carbohydrates

The seed lectin from Dioclea grandiflora and jack bean lectin concanavalin A (ConA) are both members of the Diocleinae subtribe of Leguminosae lectins. Both lectins have recently been shown to possess enhanced affinities and extended binding sites for the trisaccharide, 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present in the core region of all asparagine-linked carbohydrates (Gupta, D., Oscarson, S., Raju, S., Stanley, P. Toone, E. J. and Brewer, C. F. (1996) Eur. J. Biochem. 242, 320-326). In the present study, the binding specificities of seven other lectins from the Diocleinae subtribe have been investigated by hemagglutination inhibition and isothermal titration microcalorimetry (ITC). The lectins are from Canavalia brasiliensis, Canavalia bonariensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. Hemagglutination inhibition and ITC experiments show that all seven lectins are Man/Glc-specific and have high affinities for the core trimannoside, like ConA and D. grandiflora lectin. All seven lectins also exhibit the same pattern of binding to a series of monodeoxy analogs and a tetradeoxy analog of the trimannoside, similar to that of ConA and D. grandiflora lectin. However, C. bonariensis, C. floribunda, D. rostrata, and D. violacea, like D. grandiflora, show substantially reduced affinities for a biantennary complex carbohydrate with terminal GlcNAc residues, while C. brasiliensis, D. guianensis, and D. virgata, like ConA, exhibit affinities for the oligosaccharide comparable with that of the trimannoside. Thermodynamic data obtained by ITC indicate different energetic mechanisms of binding of the above two groups of lectins to the complex carbohydrate. The ability of the lectins to induce histamine release from rat peritoneal mast cells is shown to correlate with the relative affinities of the proteins for the biantennary carbohydrate.

GROWTH STAGE EFFECTS ON NITROGENASE, NITRATE REDUCTASE, AND UREIDE CONTENT IN NODULES OF ‘COPADA’1

Available soil nitrogen usually limits improved pasture production. Perennial, high-nitrogen-fixing forage legumes are desirable for tropical swards. The drought-tolerant Phaseoleae, ‘Copada’ (Cratylia floribunda Mart. ex. Benth.) is well adapted to a wide range of soil texture, pH. and fertility levels of Oxisol-Ultisols in the subhumid neotropics. It is highly competitive and has vigorous regrowth and high nitrogen-fixing capabilities under intensive grazing regimes. Our objective was to study interrelationships of plant growth and the characteristics of determinate mature and distinctive nonmature nodules for enhanced nitrogen fixation. Determinations included nitrogenase activity (C2H2 reduction), nodule cytosol ureide composition, and selected enzyme components associated with symbiotic nitrogen fixation. Plants were grown in the greenhouse, with ages of individual plants recorded as days from emergence at germination. Copada top growth increased from 0.34 to 2.03 grams from 24 to 62 days from emergence. Nodule fresh weight increased from 124.7 to 786.6 milligrams per plant, and nodule numbers approximately doubled, from 69.4 to 135.0. Nitrogenase activity (C2H2 reduction) increased from 28.4 to 200.9 ± mole C2H4 h-1 g-1. Total cytosol ureides of determinate mature nodules increased from 0.21 to 2.59 ± mole g-1 fresh nodule. Differential activity levels of mature nodule cytosol enzymes included increased aspartate aminotransferase (AST) 25.6 to 47.3 IU, glutamate dehydrogenase (GDH) 1.51 to 2.94 IU, and glutamine synthetase (GS) 6.71 to 20.58 IU during the 6-week period. Glutamate synthase (GOGAT) decreased from 3.99 to 1.75 IU. The principal factors influencing nitrogenase activity (C2H2 reduction) were apparent, with a highly significant regression for mature nodule cytosol as: nitrogenase activity (C2H2 reduction) = 2.57 nodular weight + 3.27 ureide + 2.67 GDH - 1.98 GOGAT, R2 = 82.6, CV = 9.24 percent. Differences were highly significant for determinate mature nodule enzyme activity levels between Copada plants during the plant growth cycle. Differences were not statistically significant for comparative composition between mature and distinctive nonmature nodule cytosol from the same Copada plant for GDH and GOGAT. AST and GS were significantly higher within mature nodule cytosol. Nitrate reductase (NR) was significantly higher, with 103.3 IU g-1 for nonmature compared with 4.5 IU g-1 for mature nodules. No significance was apparent for cytosol levels of 2-oxoglutarate (αKG), pyridoxal-5-phosphate, and pyridoxamine-5-phosphate (PLP), and total nonstructural carbohydrates (CHO) as a function of nodule maturity. Components of nonmature nodules significantly influencing cytosol nitrate reductase were determined to be NR = 2.01 AST - 2.95 nodular weight + 2.89 PLP + 1.85 ureide, R2 = 54.2, CV = 39.8 percent. Mature nodule cytosol ureide composition, 1.37 ± mol, was highly significant over 0.01 ±mol g-1 nonmature nodule. A highly significant multiple regression was determined for total cytosol ureide = 4.71 GS + 1.39 nodular weight - 1.32 AST - 1.56 αKG, R2

Caracteres culturais das bactérias dos nódulos de algumas leguminosas tropicais

23 isolates from root nodules of 13 leguminous species were tested in laboratory for cultural and physiological characteristics. The following conclusions can be drawn: 1 - The cultural and physiological characters were not enough to identify the different isolates. 2 - The data from cultural and physiological tests help the identification of the bacteria, when checked with data from cross-inoculation tests reported elsewhere (GALLI, 1958). The bacteria studied herein may be grouped as the following: a) nodule bacteria from Vigna sinensis, Calopogonium muconoides, Centrosema pubescens, Desmodium adscendens, D. discolor, Indigofera mucronata, I. subulata, I. sumatrana, Stylosanthes guyanensisA Tephrosia cândida and Teramnus uncinatus: bacteria with scanty growth; alcaline reaction in milk-brom cresol purple; no acid production in medium with any of the carbohydrates used. b) nodule bacteria from pea and from Vicia obscura: bacteria with moderate growth; alcaline reaction, with serum zone, in milk-brom cresol purple; acid production in medium with all the carbohydrates used. c) nodule bacteria from Leucaena glauca: bacteria with moderate growth; alcaline reaction, with serum zone, in milk-brom cresol purple; acid production from rafinose and manitol. d) nodule bacteria from Cratylia floribunda: bacteria with scanty growth; alcaline reaction in milk-brom cresol purple; acid production from mannose and ramnose.