Purification and partial characterization of a new mannose/glucose‐specific lectin from Dialium guineense Willd seeds that exhibits toxic effect

A new mannose/glucose‐specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b‐Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d ‐mannose, d ‐glucose, and derived sugars, especially α‐methyl‐d ‐mannopyranoside and N‐acetyl‐d ‐glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28–30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate‐binding site. Copyright © 2013 John Wiley & Sons, Ltd.     

Purification and characterization of a mannose/N-acetyl-D-glucosamine-specific lectin from the seeds of Platymiscium floribundum Vogel

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.     

Purification and molecular characterization of a novel mannose‐specific lectin from Dioclea reflexa hook seeds with inflammatory activity

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G‐50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α‐methyl‐d ‐mannoside, d ‐mannose, and d ‐glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0–7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, β, and γ) with masses of 25 562, 12 874, and 12 706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp. Copyright © 2015 John Wiley & Sons, Ltd.     

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied.MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed.ResultsThe purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (K_a=4.9 (±0.2)×10⁴ M^?1), which indicated static quenching (Stern-Volmer (SV) constant K_sv=4.6 (±0.2)×10⁴ M^?1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa.ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored.General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.     

Purification,characterization and partial sequence of a pro‐inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single‐step affinity chromatography in Sephadex® G‐50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α‐chain) and two minor components (β‐chain and γ‐chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA‐like lectin. In comparison with the average molecular mass of α‐chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D‐glucose, D‐mannose, and α‐methyl‐D‐mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH‐sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses. Copyright © 2014 John Wiley & Sons, Ltd.     

Purification and characterization of a D‐mannose specific lectin from the green marine alga,Bryopsis plumosa

A d ‐mannose specific lectin was purified from the green marine alga, Bryopsis plumosa (Huds.) Ag. The lectin agglutinated horse and sheep erythrocytes. Matrix assisted laser desorption/ionization time of flight mass spectrometry, size exclusion chromatography, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two dimensional gel electrophoresis (2DE) results showed that the lectin was a monomer with molecular weight of 17 kDa and pI 7.3. The agglutinating activity was inhibited by d ‐mannose (1 mM), α‐methyl‐D‐mannose (4 mM) and l ‐fucose (8 mM). d ‐glucose (125 mM) showed weak inhibition. The lectin did not need divalent cations for agglutinating activity. N‐terminal amino acid sequence of the lectin was analyzed. As the lectin was novel, we named it BPL‐2 (Bryopsis plumosa lectin 2). Full cDNA sequence of BPL‐2 was obtained using cDNA library. It was comprised of 624 bp of open reading frame and 167 bp/57 bp of 3′/5′ untranslated regions as well as N‐terminal signal peptide. No antimicrobial activity of BPL‐2 was observed in four bacteria strains tested.     

家蝇蛹甘露糖结合凝集素的结构及免疫调节作用

分析了一种分子量为24 kDa,具有免疫活性的新型家蝇蛹甘露糖结合凝集素(MBL-1)的结构,为深入研究其结构与功能之间的关系提供依据.首先通过凝胶电泳及Schiff's染色确定此新型家蝇蛹凝集素具有糖链结构.通过β-消除反应、红外光谱、原子力显微镜和蛋白N端测序仪对其结构进行分析.结果表明MBL-1是一种存在N-糖苷键,蛋白含量为97.20％、糖含量为2.55％,直径60～100 nm的椭球状蛋白单体,肽链N-端封闭.MTT实验证明MBL-1可以明显促进巨噬细胞的增殖且具有浓度依赖性,扫描电镜观察结果表明,MBL-1作用后的巨噬细胞形态呈现活化状态.可见,分离纯化出的MBL-1是一种具有明显的免疫调节活性的新型凝集素,为开发天然免疫增强剂和进一步分析其结构及其作用机制提供了参考.     

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.     

Purification and properties of a lectin from ascomycete mushroom,Ciborinia camelliae

A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-D-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of approximately 17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found.     

A cytopathic effect of Trichomonas vaginalis probably mediated by a mannose/N-acetyl-glucosamine binding lectin.

The pathogenic effect of a highly pathogenic strain of Trichomonas vaginalis on McCoy cell monolayers was investigated. Specific inhibition of the cytopathic effect by monosaccharides, such as N-acetyl-glucosamine (GlcNAc) and mannose (Man), was observed. Our preliminary results suggest that the pathogenicity of T. vaginalis depends on a lectin specifically sensitive to GlcNAc and to a lesser extent to Man. Although N-acetyl-mannosamine was found to be the most efficient inhibitor, this effect seems to be unrelated to the natural biological behaviour of the infested host.     

Purification and partial characterization of a novel lectin from Dioclea lasiocarpa Mart seeds with vasodilator effects

A lectin from seeds of Dioclea lasiocarpa (DLL) was purified in a single step by affinity chromatography in a Sephadex G‐50 column. DLL haemagglutinated rabbit erythrocytes showing stability even after 1 h of exposure to a different pH values (optimal between pH 6.0 and 8.0) but was inhibited after incubation with d ‐mannose and d ‐glucose. The pure protein possessed a molecular weight of 25 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 25,410Da by mass spectrometry. The results analyzed by the software SELCON 3 indicate that β‐sheet secondary structures are predominant in DLL (approximately 40.2% antiparallel β‐sheet, 4.6% parallel β‐sheet, 7.2% α‐helices, 17.3% turns, and 28.7% unordered structures). Mechanical activity of isolated aorta from rat measured by cumulative concentration curves of DLL, performed at the contraction plateau induced by phenylephrine in either endothelium‐intact or denuded aorta. DLL (IC₅₀ = 34.12 ± 3.46 µg/ml) relaxed precontracted endothelized aortic rings by 34.61 ± 9.06%, 55.19 ± 11.9%, and 81.33 ± 14.35%, respectively, at 10 µg/ml (initial concentration), 30 µg/ml, and 100 µg/ml (maximum effect). All effects occurred via interaction with lectin domains and participation of nitric oxide. Copyright © 2012 John Wiley & Sons, Ltd.     

The galactose‐binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines

In this study, the amino acid sequence and anti‐inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% – 3 h, 19% – 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF‐α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate‐binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF‐α (68%) and IL1‐β (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti‐inflammatory activity by inhibiting neutrophil migration by reducing TNF‐α and IL1‐β levels via the lectin domain. Copyright © 2015 John Wiley & Sons, Ltd.     

Purification,primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.     

Isolation and characterization of a lectin from the seeds of Erythrina Edulis

A galactose-specific lectin was isolated from the seeds of Erythrina edulis. The protein was purified by affinity chromatography of the globulin fraction on an allyl-galactoside polyacrylamide gel. The hemagglutination properties, amino acid composition, A₂₈₀, MW of the protein and of its subunits, carbohydrate content, electrophoretic pattern and isoelectric point were determined. Comparison of its properties with those of other Erythrina lectins shows that the protein is a distinct member of this group of lectins.     

Purification and primary structure determination of a galactose-specific lectin from Vatairea guianensis Aublet seeds that exhibits vasorelaxant effect

Vatairea guianensis seeds, a typical plant from the Brazilian Amazon region that belongs to the Dalbergieae tribe, possess a lectin that was isolated by precipitation with solid ammonium sulfate followed by guar gum affinity chromatography. This lectin was named VGL. The V. guianensis lectin strongly agglutinated rabbit erythrocytes and was inhibited by d-galactose and d-galactose-derived sugars, especially N-acetyl-d-galactosamine. VGL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with ethylenediamine tetraacetic acid (EDTA). In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the purified VGL exhibited an electrophoretic profile consisting of a major 30–32 kDa double band, which is termed the alpha-chain, and two minor components of 18 and 15 kDa, which are referred to as the beta- and gamma-chains, respectively. An analysis using electrospray ionization mass spectrometry also indicated that purified VGL contains a mixture of chains with molecular weights of 28,437 ± 2, 14,952 ± 2 and 12,332 ± 2. The complete amino acid sequence of VGL, as determined using tandem mass spectrometry, consists of 239 amino acid residues. VGL is a glycoprotein exhibiting high similarity in primary structure to other lectins from evolutionarily related plants, such as Vatairea macrocarpa lectin and lectins belonging to the Sophoreae tribe. VGL exhibits vasorelaxant activity in contracted rat aortas, an effect that is strictly dependent on the endothelium and involves nitric oxide and the lectin domain.     

Purification and molecular cloning of a new galactose-specific lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> seeds

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.     

Carbohydrate recognition by the rhamnose‐binding lectin SUL‐I with a novel three‐domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL‐I, which is a rhamnose‐binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL‐I (rSUL‐I) was produced in Escherichia coli cells, and its carbohydrate‐binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose‐terminated N‐glycans. rSUL‐I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three‐dimensional structure of the rSUL‐I/l ‐rhamnose complex was determined by X‐ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL‐I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l ‐rhamnose molecules are mainly recognized by rSUL‐I through hydrogen bonds between its 2‐, 3‐, and 4‐hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL‐I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.     

Purification, properties, and crystallographic data for a principal nontoxic lectin from seeds of Abrus precatorius.

Nontoxic abrus lectin has been prepared by a new purification procedure. The method is accomplished by 45% saturation ammonium sulfate fractionation from a 5% acetic acid extract of the seeds of Abrus precatorius followed by diethylaminoethyl-Sephadex A-50 and Sepharose 4B affinity chromatography. The abrus lectin appeared homogeneous as judged by electrophoresis, analytical ultracentrifugation, and isoelectric focusing. The lectin molecule has a weight of 126,000 as determined by sedimentation equilibrium. It is composed of four subunits, of which two pairs have either identical or closely similar molecular sizes (33,800 and 32,200 daltons) as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of amino acid analyses are given; none of the cysteic acid appears to arise from cysteine. An electrofocusing experiment indicated the isoelectric point to be 5.0. Crystals large enough for x-ray investigation were obtained by a vapor diffusion technique. X-ray precession photographs revealed that abrus lectin crystallizes in a tetragonal unit cell of symmetry P41212 and dimensions a = 140 and c = 210 A. The asymmetric unit contains 2 protein molecules of molecular weight 126,000 and has a solvent content of approximately 41% by volume.