Research Article Received: 18 September 2013,
Revised: 05 November 2013,
Accepted: 08 November 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2340
Purification, characterization and partial sequence of a pro-inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams Mayara Q. Santiagoa, Cintia C. F. Leitãoa, Francisco N. Pereira-Juniora, Vanir R. Pinto-Juniora, Vinicius J. S. Osternea, Claudia F. Lossioa, João B. Cajazeirasa, Helton C. Silvaa, Francisco V. S. Arrudac, Livia P. Pereirad, Ana M. S. Assreuyd, Kyria S. Nascimentoa, Celso S. Naganob and Benildo S. Cavadaa* 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. Keywords: plant lectin; Canavalia oxyphylla; ESI mass spectrometry; toxic effect; pro-inflammatory
INTRODUCTION
J. Mol. Recognit. 2014; 27: 117–123
a M. Q. Santiago, C. C. F. Leitão, F. N. Pereira-Junior, V. R. Pinto-Junior, V. J. S. Osterne, B. S. Cavada Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza, CE, 60440-970, Brazil b C. S. Nagano Laboratório de Espectrometria de Massa aplicado a Proteínas (LEMAP), Federal University of Ceará, Av. Humberto Monte s/n, Bloco 825, Campus do Pici, Fortaleza, CE, 60440-970, Brazil c F. V. S. Arruda Laboratório Integrado de Biomoléculas (LIBS), Integrated Laboratory of Biomolecules (LIBS), Department of Pathology and Legal Medicine, Federal University of Ceará, Rua Monsenhor Furtado, S/N—Rodolfo Teófilo, Fortaleza, CE, 60441-750, Brazil d L. P. Pereira, A. M. S. Assreuy Laboratório de Fisiofarmacologia da Inflamação (LAFFIM), Institute of Biomedical Sciences, State University of Ceara, Av Paranjana 1700, 60740-000, Fortaleza, Ceará, Brazil
Copyright © 2014 John Wiley & Sons, Ltd.
117
Lectins are defined as a structurally heterogeneous group of proteins or glycoproteins that possess at least one noncatalytic domain that binds reversibly to a specific monosaccharide or oligosaccharide (Peumans and Van Damme, 1995a, 1995b). Lectins have been studied extensively in recent years based on their overall utility as molecular tools involving (i) cell surface composition, growth, and differentiation; (ii) pathological mechanisms; and (iii) the isolation and characterization of glycoconjugates (Sharon and Lis, 2004; Sharon, 2007). Legume lectins constitute the best-studied group of plant lectins, and hundreds of them have been isolated and characterized in relation to their chemical, physicochemical, structural, and biological properties (Van Damme et al., 1998; Peumans and Van Damme, 1995a, 1995b). Specifically, legume lectins are normally composed of two or four monomers, presenting a molecular mass of about 25–30 kDa, with each monomer presenting a unique, highly conserved carbohydrate binding site, as well as conserved metal binding sites for divalent cations (calcium and
* Correspondence to: Benildo S. Cavada, Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza, CE 60440-970, Brazil. E-mail: [email protected]
M. Q. SANTIAGO ET AL. manganese). The monomers are associated by noncovalent interactions (Brinda et al., 2004). Lectins of the Diocleinae subtribe are highly homologous with regard to carbohydrate binding specificities and biochemical and physicochemical properties, but they differ in biological activity (Barral-Netto et al., 1992; Assreuy et al., 2009). In general, Diocleinae lectins elicit anti-inflammatory or pro-inflammatory responses, depending on the administration route used, being inhibitory when administered systemically, but stimulatory when applied locally. ConBr, a Diocleinae lectin extracted from Canavalia brasiliensis, is an exception, producing only proinflammatory responses (Assreuy et al., 1997). Minor differences in the ratios of dimeric and tetrameric forms in the lectins, together with differences in the relative orientations of the carbohydrate-binding sites in the quaternary structures, have been hypothesized to contribute to the differences in biological activities exhibited by Diocleinae lectins (Cavada et al., 1994). The genus Canavalia comprises 51 species of leguminous vines, which are widespread in Brazil, Guiana, and the Atlantic coastal regions of tropical Central America and Mexico (Lackey, 1981). In the present study, we isolated a glucose/mannosebinding lectin [Canavalia oxyphylla Lectin (CoxyL)] from the seeds of Canavalia oxyphylla Standl. & L. O. Williams, a woody vine of the Diocleinae subtribe. The purified lectin was physicochemically characterized, in particular, its toxic effect on Artemia sp. nauplii, the brine shrimp, and its pro-inflammatory activity.
Hemagglutination activity and sugar specificity The hemagglutination assay was performed in microtiter plates by serial dilution using 3% native rabbit erythrocytes treated with the proteolytic enzymes trypsin and papain (Ainouz et al., 1992). The carbohydrate binding specificity of CoxyL was determined by the ability of different sugars and glycoproteins to inhibit erythrocyte agglutination (Ramos et al., 1996). Serial dilution was performed on sugars/glycoproteins (100 mM/1 mg/ml initial concentrations, respectively, and CoxyL was subsequently added to each dilution at a concentration of 4 hemagglutinating unit [HU]). HU was expressed as the value of the highest dilution giving positive hemagglutination per milliliter of sample, and inhibition values were expressed by minimum inhibitory concentration. Effects of pH, temperature, and divalent ions on lectin-induced hemagglutination
MATERIAL AND METHODS Purification of Canavalia oxyphylla lectin The seeds obtained were peeled and ground in a coffee grinder (CadenceTM MDR301 Monovolt). After seed flour obtained were stored in an oven at a temperature of 37°C, dry flour was obtained. Soluble proteins were extracted in 150 mM NaCl [1:10 (w: v)] under constant stirring for 4 h at room temperature. Subsequently, the extract was centrifuged at 10 000 × g at 4°C for 20 min, and the supernatant was filtered on filter paper porosity 6 μm (WhatmanTM). The resulting supernatant was applied to Sephadex® G-50 affinity column (6.5 × 1.5 cm) previously equilibrated with 150 mM NaCl. Unbound material (P1) was washed with the same solution, and lectin was eluted with 100 mM D-glucose in the equilibrium solution. To estimate protein concentration, the eluted fractions were monitored by absorbance at 280 nm and by Bradford (1976), using bovine serum albumin as standard. The eluate was collected, dialyzed against distilled water, and lyophilized. The homogeneity of the sample was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the pure protein was used for characterization tests. Electrophoretic analysis
118
The purity and apparent molecular weight of the lectin were determined by PAGE in the presence of SDS (SDS-PAGE). The experiment was performed in a discontinuous buffer system as described by Laemmli (1970) using 15% total concentration polyacrylamide as the separating gel and 4% total concentration polyacrylamide as the stacking gel. The procedure was carried out using a Mini Protean II apparatus (BioRad; Milan; Italy) under constant current of 25 mA for 60 min. At the end of electrophoresis, the separated protein bands were stained with Coomassie
wileyonlinelibrary.com/journal/jmr
Brilliant Blue R-250. The following molecular markers were used: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). The presence of disulfide bonds was tested by addition of 2% β-mercaptoethanol in the sample prior to the electrophoretic run. The presence of covalently bound carbohydrate was evaluated by staining with periodic acid-Schiff’s reagent after SDS-PAGE (Zacharius et al., 1969).
The pH stability of CoxyL was determined by dissolving the lectin in different buffers, all containing 150 mM NaCl, and measuring the hemagglutinating activity of the solutions. The buffers used in this test were 100 mM sodium citrate for pH 4.0 and 6.0, 100 mM sodium acetate for pH 5.0, 100 mM sodium phosphate for pH 7.0, 100 mM Tris–HCl for pH 8.0, and 100 mM GlycineNaOH for pH 9.0 and 10.0. To investigate the thermal stability, CoxyL (0.125 mg/ml) was prepared in 150 mM NaCl and incubated at various temperatures (40, 50, 60, 70, 80, 90, and 100°C) for 60 min. After incubation, the solutions were cooled to room temperature, and their hemagglutinating activity was determined. For analysis of the influence of divalent cations on the lectin hemagglutinating activity, CoxyL was dialyzed against 25 mM ethylenediaminetetraacetic acid (EDTA) containing 150 mM NaCl for 24 h at 4°C, followed by an additional dialysis against 150 mM NaCl. HU of treated and untreated samples were compared. Molecular mass determination The average molecular mass of C. oxyphylla lectin was determined by electrospray ionization mass spectrometry using a hybrid quadrupole/ion mobility separator/orthogonal acceleration time-of-flight mass spectrometer (SYNAPT HDMS System, Waters Corp., Milford, MA, USA). C. oxyphylla lectin ( ρmol/μl in 50% acetonitrile with 0.1% formic acid) was infused into the system at 0.5 μl/min. The capillary and cone voltages were set at 3.5 kV and 50 V, respectively. The source temperature was maintained at 80°C, and nitrogen was used as a drying gas (flow rate of 100 l/h). The instrument was calibrated with fragments of the double protonated ion of [Glu1]-fibrinopeptide B (SigmaAldrich, St. Louis, MO, USA) (m/z 785.84). Data acquisition was performed at m/z in the range of 800–3000, using MASS LYNX 4.0 software (Waters), and the spectra of multiply charged
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL ions were deconvoluted using maximum entropy techniques (Ferrige et al., 1992).
of research animals (National Institute of Health guidelines) and approved by the Institutional Animal Care and Use Committee of the State University of Ceará (UECE–N° 0559924–4).
Proteolytic cleavages Protein digestion was performed as previously described by Shevchenko et al. (2006). To accomplish this, C. oxyphylla lectin was submitted to SDS-PAGE, and the bands were excised and bleached with 50 mM ammonium bicarbonate in 50% acetonitrile. Bands were dehydrated in 100% acetonitrile and dried in a Speedvac (LabConco, Kansas City, MO, USA). For proteolytic cleavage, gels were rehydrated in an enzyme solution of 50 mM ammonium bicarbonate containing trypsin (Promega, Madison, WI, USA) at 37°C overnight. The obtained peptides were extracted in a solution of 50% acetonitrile with 5% formic acid and concentrated in Speedvac.
Effect of CoxyL on rat paw edema Paw edema was measured immediately before (zero time) intraplantar subcutaneous (s.c.) injection of CoxyL (0.01, 0.1, and 1 mg/Kg) and thereafter (30–600 min) using a hydroplethysmometer (PanLab, Barcelona, Spain). Results were expressed as the increase in paw volume (ml) calculated by subtracting the basal volume measured at zero time or area under curve (AUC) (arbitrary units) (Landucci et al., 1995). Data are presented as mean ± S.E.M. The statistical differences, set at p < 0.05, were analyzed using analysis of variance followed by Bonferroni’s test.
Tandem mass spectrometry analysis The peptides obtained in the different proteolytic cleavages were separated on a BEH300 C18 column (100 × 100 mm) using a nanoAcquity™ system and eluted with acetonitrile gradient (10–85%), containing 0.1% formic acid at 600 μl/min. The liquid chromatography system was connected to a nanoelectrospray mass spectrometer source (SYNAPT HDMS System, Waters Corp., Milford, USA). The mass spectrometer was operated in positive mode, using a source temperature of 90°C and capillary voltage of 3.5 kV. The lock mass used in acquisition was m/z 785.84 ions of the [Glu1] fibrinopeptide B. The LC-MS/MS experiment was performed according to the data dependent acquisition function, selecting MS/MS doubly or triply charged precursor ions, which were fragmented by collision-induced dissociation (CID) using argon as collision gas and ramp collision energy that varied according to the charge state of the selected precursor ion. The CID spectra were interpreted manually using the Peptide Sequencing tool from MASS LYNX 4.0 software (Waters). The C. oxyphylla lectin peptide sequences were compared with all non-redundant proteins deposited in the National Center of Biotechnology Information using BLAST (Altschul et al., 1997), and the proteins with the best e-value were selected to sequence alignments at ClustalW (Gasteiger et al., 2005).
RESULTS AND DISCUSSION Purification of CoxyL and hemagglutination assays The crude extract of C. oxyphylla seeds showed strong agglutination activity in native rabbit erythrocytes or treated with trypsin and papain. The inhibition assay of hemagglutinating activity with carbohydrates showed that the lectin has specificity for D-glucose, D-mannose, and α-methyl-D-mannoside. Glycans present in ovalbumin and fetuin were able to inhibit the hemagglutinating activity of CoxyL, perhaps by the presence of mannose in its composition (Table 1). The specificity profile of CoxyL is similar to that of other lectins in the Canavalia genus. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column, in which the lectin was quantitatively retained in the gel and eluted with 100 mM D-glucose (Figure 1A). The soluble protein content and the specific activity of the crude lectin extract were 10.8 mg/ml and 95 HU/mg protein, respectively. For the purified lectin, the values were 0.82 mg/ml and 2498 HU/mg protein, respectively. The specific activity increased by 26.3-fold in the pure lectin (Table 2).
Artemia lethality test CoxyL (200 μg/ml) was homogenized with artificial sea water and assayed in triplicate on Linbro plates 24 wells, containing 10 specimes of Artemia sp. nauplii per well with a final volume of 2 ml. Lectin solution was added to the wells to reach concentrations of 12.5, 25, 50, and 100 μg/ml compared with controls containing sea water only. To block the activity of the lectin, CoxyL (12.5, 25, 50, and 100 μg/ml) was incubated in artificial sea water containing 100 mM of mannose for 1 h at 37°C. After 24 h, the percentage of deaths and LC50 values were obtained using the simple Microsoft Excel 2013 and calculated by probit analysis as described by Finney (1971). Pro-inflammatory activity Animals
J. Mol. Recognit. 2014; 27: 117–123
Carbohydrates and glucoproteins D-glucose D-mannose D-galactose N-acetyl-D-glucosamine α-methyl-D-mannoside α-methyl-D-galactoside α-lactose β-lactose Ovalbumin Fetuin
MIC 50 mM 25 mM NI NI 12.5 mM NI NI NI 0.25 mg/ml 0.125 mg/ml
MIC, minimum inhibitory concentration; NI, sugar not inhibitory until a concentration of 100 mM is reached.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
119
Male Wistar rats (150–250 g), n = 6–7 animals per group, were maintained in a controlled 12/12 h light/dark cycle, at 25°C with free access to food and water. Experiments were conducted in accordance with current guiding principles for the care and use
Table 1. Inhibitory effect of saccharides and glycoproteins on the hemagglutinating activity of Canavalia oxyphylla lectin
M. Q. SANTIAGO ET AL. Effects of pH, temperature, and divalent ions on lectin-induced hemagglutination The effect of temperature on CoxyL activity is shown in Figure 2A. The lectin was shown to be thermostable, exhibiting full activity up to 60°C, with a considerable loss in activity occurring at 70°C (loss of 87.5%), and no hemagglutinating activity was detected at 90°C. Examination of CoxyL activity toward different pH values showed that the lectin was pH-sensitive. CoxyL exhibited maximum activity at pH 7.0, indicating that the lectin is more stable in this condition, while in acidic pH (4.0–6.0), the lectin showed low stability with only 25% of its hemagglutinating activity remaining. In basic pH (8.0–9.0), the lectin lost 50% of its activity in pH 8.0, and a further decrease in pH beyond 9.0 caused a 75% loss in hemagglutination activity in pH 9.0 (Figure 2B). The thermostability and pH stability of CoxyL are similar in related Diocleinae lectins (Moreira et al., 1995; Correia et al., 2011). After treating the lectin with EDTA, the hemagglutination activity of CoxyL remained unchanged. These data suggest that CoxyL, in contrast to various other leguminous lectins (Cavada et al., 2001; Loris et al., 1998), does not need divalent cations for its activity, as they are most probably tightly bound to the protein. Although this property is especially rare in the case of lectins from the Diocleinae subtribe, the hemagglutinating activity of other Diocleinae lectins, such as those from Canavalia cathartica, was also unaffected by EDTA (Suseelan et al., 2007).
Mass spectrometry analysis Figure 1. CoxyL purification and molecular mass estimation. (A) Elution profile of Sephadex® G-50 affinity chromatography. Approximately 10 ml of the crude extract was applied to the Sephadex® G-50 column (6.5 × 1.5 cm) that was equilibrated with 150 mM NaCl. The lectin was eluted with 100 mM D-glucose at a flow rate of 1 ml/min. Fractions (approximately 1.5 ml) were collected and monitored for protein content by measuring the absorbance at 280 nm. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lane 1: molecular mass markers (phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; trypsin inhibitor and a-lactalbumin, 14.4 kDa); lane 2: Crude extract of C. oxyphylla (15 μg); lane 3: CoxyL (15 μg).
Electrophoretic analysis and presence of covalently bound carbohydrate The electrophoretic profile obtained with SDS-PAGE of the affinity-purified CoxyL, both in the presence and absence of β-mercaptoethanol, revealed a major band of 30 kDa (α-chain) (Figure 1B). The β-fragments and γ-fragments were not observed on SDS-PAGE a typical ConA-like lectin, perhaps because of low concentration of these fragments in the purified sample.
Electrospray ionization mass spectrometry confirmed that CoxyL consists of a combination of chains weighing 25 696 ± 2 Da (α-chain), 12 980 ± 2 Da (β-chain), and 12 733 ± 2 Da (γ-chain) (data not shown), suggesting that C. oxyphylla lectin is a typical ConA-like lectin subject to the posttranslational processing of circular permutation described by Carrington et al. (1985). Thus, CoxyL should be expressed as a pre-pro-protein (Ntermsignal peptide + γ-chain + linker peptide + β-chain + Ctermsignal peptide) cleaved into a γ product and a β product. The active protein is a final fused product (α-chain) with the two fragments in inverse order and having no signal or linker peptides (Cunningham et al., 1979; Carrington et al., 1985; Chrispeels et al., 1986). The β-fragment and γ-fragment observed on SDS-PAGE are unlinked products of this process, while the α-chain is the mature protein. CoxyL showed no staining by periodic acid-Schiff, suggesting that it is not a glycoprotein. The partial amino acid sequence of CoxyL was determined by tandem mass spectrometry after proteolysis of the protein band excised from an SDS-PAGE gel. Six peptides were sequenced, resulting in a total of 107 amino acid residues (Table 3). In
Table 2. Purification of lectin from Canavalia oxyphylla seeds Fraction
a
Total protein (mg/ml)
Crude extract PII (Sephadex® G-50)
10.8 0.82
b
Total HU
c
Specific activity (HU/ml)
10
2 211
95 2498
Purification (fold) 1 26.3
a
Protein content. Hemagglutinating activity expressed in hemagglutinating units (HU). c Specific activity calculated as the ratio between hemagglutinating activity and protein content. b
120
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL wilsonii (DwL) (SwissProt accession code: P86624), and Cratylia floribunda (CFL). In relation to ConA, the partial amino acid sequence of CoxyL showed differences in seven positions (ConA/CoxyL): A50V and D59G in T3; S96T in T1; H121A, M129T, and S134T in T4; and E155Q in T5. Legume lectins of the Diocleinae subtribe present similar carbohydrate-binding specificities and reveal considerable homology in amino acid sequence and tertiary structure (Calvete et al., 1999; Cavada et al., 2001). Nevertheless, Diocleinae lectins may differ substantially in biological activities (Barral-Netto et al., 1992; Gadelha et al., 2005; Assreuy et al., 2009). These differences can be associated with the position of amino acids in the carbohydrate-binding site or residues located in key positions of the quaternary structure (Calvete et al., 1999; Wah et al., 2001; Brinda et al., 2004; Nagano et al., 2008). Lethality test
Figure 2. Physicochemical properties of the CoxyL. Hemagglutinating activity of the PFL after different treatments by temperatures (A) and pH measures (B).
comparison with average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. Figure 3 shows the CID spectra of peptide T3, in which sequence-specific y-ions were used for sequence determination. Comparisons with other sequences from the National Center of Biotechnology Information databank revealed that the CoxyL peptides were similar to other Diocleinae lectins, such as Canavalia ensiformis (ConA) (SwissProt accession code: P02866), Canavalia brasiliensis (ConBr) (SwissProt accession code: P55915), Canavalia gladiata (CGL) (PDB accession code: 2EF6), Canavalia maritima (CML) (PBD accession code: 2P34), Dioclea guianensis (Dgui) (SwissProt accession code: P81637), Dioclea grandiflora (DGL) (SwissProt accession code: A9J251), Dioclea
CoxyL has toxic activity against brine shrimp (Artemia sp. nauplii), exhibiting LC50 value of 44.31 μg/ml. This effect proved to be dose dependent and was observed in a concentration range from 12.5 to 100 μg/ml (Figure 4A). The Artemia lethality test (Persoone, 1980; Sorgeloos et al., 1978) has been successfully used to determine the toxicity of biological molecules that have a variety of pharmacological activities, including anticancer agents, antivirals, insecticides, pesticides, and anti-HIV compounds (Carballo et al., 2002; Pervin et al., 2006; Ho et al., 2007). Previous work showed that other lectins, such as ConA-like, also have toxic effect (Santos et al., 2010), with LC50 between 2.52 and 15.5 μg/ml. In comparison to similar lectins, CoxyL exhibited low toxicity against Artemia. The lethality of animals abolished when the lectin was incubated with D-mannose (Figure 4B), showing that the carbohydrate recognition domain is responsible for the toxic activity against Artemia sp. Canavalia oxyphylla lectin presents edematogenic effect CoxyL induced an increase in paw edema at 0.01 mg/kg (116.7 ± 22.46 AUC), 0.1 mg/kg (138.6 ± 26.00 AUC), and 1 mg/kg (349.5 ± 28.39 AUC) compared with the saline group (18.3 ± 5.73 AUC). At the highest dose (1 mg/kg), local injection of CoxyL evoked significant paw edema in the initial 30 min, that lasted until 540 min (0.1 ± 0.03 ml), with maximal activity at 120 min (1.1 ± 0.05 ml) compared with saline (0.08 ± 0.03 ml) (Figure 5). The edematogenic effect elicited by CoxyL was characteristic of acute inflammatory responses. Similar responses had been
Table 3. Amino acid peptide sequences of CoxyL obtained using tandem mass spectrometry and their respective molecular masses Peptide* T1 T2 T3 T4 T5 T6
Experimental mass (Da)
Theoretical mass (Da)
Sequence**
1108.6128 1512.7460 1385.7667 2175.0127 2545.0549 3293.6091
1108.5044 1512.7244 1385.7065 2175.9165 2545.9644 3293.5564
VGXSATTGXYK ETNTXXSWS FTSK VGTVHXXYNSVGK SNSTAETNAXHFTFNQFTK DXXXQGDATTGTDGNLQXTR XSAVVSYPNADSATVSYDVDXDNVXPEWVR
J. Mol. Recognit. 2014; 27: 117–123
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
121
*Peptide obtained from cleavage with trypsin (T). **In peptide sequence, X represents residues of leucine or isoleucine, which cannot be distinguished by mass.
M. Q. SANTIAGO ET AL.
Figure 3. Peptide from CoxyL. Collision-induced dissociation of doubly charged ion T2 at m/z 694.39 with sequence-specific y-ion series interpreted as VGTVHXXYNSVGK.
Figure 4. Toxicity to the Artemia sp. nauplii assays. (A) Toxic effect of CoxyL at different concentrations (12.5, 25, 50, or 100 mg/ml) on Artemia nauplii; (B) toxic effect of CoxyL (100 mg/ml), CoxyL (100 mg/ml) previously incubated with 100 mM mannose. Mean_SEM (n = 5). *p < 0.05 compared with control.
122
previously shown for the lectin isolated from Canavalia maritma seeds, in which the edematogenic effect lasted 300 min, while the edematogenic effect induced by ConBr and CGL was more lasting, about 32 h, compared with CoxyL (Assreuy et al., 2009). These data are in line with other reports that highlights the relation structure and biological activities of these homologous lectins (Barral-Netto et al., 1992; Calvete et al., 1999; Gadelha et al., 2005; Cavada et al., 2011).
wileyonlinelibrary.com/journal/jmr
Figure 5. Edematogenic effect of the lectin Canavalia oxyphylla lectin. (A) Paw edema (ml) induced by C. oxyphylla. Animals received CoxyL (0.01; 0.1, and 1 mg/Kg, s.c.) or saline (0.01 ml per paw, s.c.). Edema was measured immediately before (zero time) and thereafter (30–600 min) using a hydroplethysmometery and expressed as (A) variation in paw volume displacement (ml) or (B) area under curve between different times and time zero. Mean ± E.P.M. (n = 6). Analysis of variance and Bonferroni’s test. *p < 0.05 compared with saline.
CONCLUSION CoxyL showed a combination of chains weighing 25 696 ± 2 Da (α-chain), 12 980 ±2 Da (β-chain), and 12 733 ±2 Da (γ-chain).
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL It was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). CoxyL demonstrated toxicity against Artemia sp. nauplii with dose-dependent effect, and it showed pro-inflammatory activity in rats by inducing an increase in paw edema. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.
Acknowledgements This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP). A. M. S. A., K. S. N., C. S. N., and B. S. C are senior investigators of CNPq, and David Martin helped with the English editing of the manuscript.
REFERENCES
J. Mol. Recognit. 2014; 27: 117–123
ExPASy server. In The proteomics protocols handbook, Walker JM (ed). Humana Press: Totowa NJ USA; 571–607. Ho JC, Chen CM, Row LC. 2007. Oleanane-type triterpenes from the flowers, pith, leaves, and fruit of Tetrapanax papyriferus. Phytochemistry 68: 631–635. Lackey JA. 1981. Tribe 10. Phaseoleae DC. (1825), In Advances in legume systematics. Part 1, Polhill RM, Raven PH (eds). Royal Botanic Gardens, Kew: UK, 301–327. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680–685. Landucci ECT, Antunes E, Donato JL, Faro RRA, Hyslop S, Marangoni S, Oliveira B, de Nucci G. 1995. Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2. Brit. J. Pharm. 114: 578–583. Loris R, Hamelryck T, Bouckaert J, Wyns L. 1998. Legume lectin structure. Biochim. Biophys. Acta 1383: 9–36. Moreira RA, Cordeiro EF, Cavada BS, Nunes EP, Fernandes AG, Oliveira JTA. 1995. Lectins and the chemotaxonomy of the genus Diocleinae (Leguminosae-Phaseoleae). R. Bras. Fis. Veg. 7(1): 7–14. Nagano CS, Calvete JJ, Barettino D, Pe´rez A, Cavada BS, Sanz L. 2008. Insights in to the structural basis of the pH-dependent dimertetramer Equilibrium through crystallographic analysis of recombinant Diocleinae lectins. Biochem. J. 409: 417–428. Persoone G. 1980. Proceeding of the international symposium on brine shrimp Artemia salina. University Press: Belgim; 1–3. Pervin F, Hossain MM, Khatun S, Siddique SP, Salam KA, Karim MR, Absar N. 2006. Comparative citotoxicity study of six bioactive lectins purified from pondweed (Potamogeton nodosus Poir) rootstock on Brine Shrimp. J. Med. Sci. 6: 999–1002. Peumans WJ, Van Damme EJM. 1995a. Lectins as plant defense proteins. Plant Physiol. 109: 347–352. Peumans WJ, Van Damme EJM. 1995b. The role of lectins in plant defense. Histochem. J. 27: 253–271. Ramos MV, Moreira RA, Cavada BS, Oliveira JTA, Rouge P. 1996. Interaction of lectins from the sub tribe Diocleinae with specific ligands. R. Bras. Fisiol. Veg. 8: 193–199. Santos AF, Cavada BS, Rocha BAM, Nascimento KS, Sant’Ana AEG. 2010. Toxicity of some glucose/mannose-binding lectins to Biomphalaria glabrata and Artemia salina. Bioresour. Technol. 101: 794–798. Sharon N. 2007. Lectins: carbohydrate-specific reagents and biological recognition molecules. J. Biol. Chem. 282: 2753–2764. Sharon N, Lis H. 2004. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14: 53–62. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. 2006. In gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1: 2856–2860. Sorgeloos P, Van Der Wielen CR, Persoone G. 1978. The use of artemia nauplii for toxicity tests—a critical analysis. Ecotoxicol. Environ. Saf. 2: 249–255. Suseelan KN, Bhagwath A, Pandey R, Gopalakrishna T. 2007. Characterization of Con C a lectin from Canavalia cathartica Thouars seeds. Food Chem. 104(2): 528–535. Van Damme EJM, Peumans WJ, Barre A, Rougé P. 1998. A novel family of lectins evolutionarily related to class V Chitinases: an example of neofunctionalization in legumes. Crit. Rev. Plant Sci. 17: 575–692. Wah DA, Romero A, Gallego del Sol F, Cavada BS, Ramos MV, Grangeiro TB, Sampaio AH, Calvete JJ. 2001. Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association. J. Mol. Biol. 310: 885–894. Zacharius RM, Zell TE, Morrison JH, Woodlock JJ. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30(1):148–152.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
123
Ainouz IL, Sampaio AH, Benevides NMB, Freitas ALP, Costa FHF, Carvalho MR, Joventino FP. 1992. Agglutination of enzyme treated erythrocytes by Brazilian marine algae extracts. Botânica Marinha 35: 447–479. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17): 3389–402. Assreuy AMS, Shibuya MD, Martins GJ, Souza MLP, Cavada BS, Moreira RA, Oliveira JTA, Ribeiro RA, Flores CA. 1997. Anti-inflammatory effect of glucose-mannose binding lectins isolated from Brazilian beans. Mediat. Inflamm. 6: 201. Assreuy AMS, Fontenele SR, Pires AF, Fernandes DC, Rodrigues NV, Bezerra EH, Moura TR, Nascimento KS, Cavada BS. 2009. Vasodilator effects of Diocleinae lectins from the Canavalia genus. N-S Arch. Pharmakol. 380: 509. Barral-Netto M, Santos SB, Barral A, Moreira LIM, Santos CF, Moreira RA, Oliveira JTA, Cavada BS. 1992. Human lymphocyte stimulation by legume lectins from the Diocleae tribe. Immunol. Invest. 21: 297. Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein-dye binding. Anal. Biochem. 72: 248–254. Brinda KV, Mitra N, Surolia A, Vishveshwara S. 2004. Determinants of quaternary association in legume lectins, Protein Sci. 13: 1735–1749. Calvete JJ, Thole HH, Raida M, Urbanke C, Romero A, Grangeiro TB, Ramos MV, Rocha IMA, Guimarães FN, Cavada BS. 1999. Molecular characterization and crystallization of Diocleinae lectins. Biochim. Biophys. Acta 1430: 367–375. Carballo JL, Hernández-Inda ZL, Pérez P, García-Grávalos MD. 2002. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnol. 2: 17–21. Carrington DM, Auffret A, Hanke DE. 1985. Polypeptide ligation occurs during post-translational modification of Concanavalin A. Nature 313: 64–67. Cavada BS, Grangeiro TB, Ramos MV, Crisostomo CV, Silva LM, Moreira RA, Oliveira JTA. 1994. Lectin from Dioclea guianensis var. lasiophylla Duke seeds mobilization during germination and seedlings growth in the dark. Rev. Bras. Fisiol. Veg. 6: 21–25. Cavada BS, Barbosa T, Arruda S, Grangeiro TB, Barral Netto M. 2001. Revisiting proteus: do minor changes in lectin structure matter in biological activity? Lessons from and potential biotechnological uses of the Diocleinae subtribe lectins. Curr. Protein Pept. Sci. 2: 123–135. Chrispeels MJ, Hartl PM, Sturm A, Faye L. 1986. Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. J. Biol. Chem. 261: 10021–10024. Correia JL, do Nascimento AS, Cajazeiras JB, Gondim AC, Pereira RI, de Sousa BL, da Silva AL, Garcia W, Teixeira EH, do Nascimento KS, da Rocha BA, Nagano CS, Sampaio AH, Cavada BS. 2011. Molecular characterization and tandem mass spectrometry of the lectin extracted from the seeds of Dioclea sclerocarpa Ducke. Molecules 16: 9077–9089. Cunningham BA, Hemperly JJ, Hopp TP, Edelman GM. 1979. Favin versus concanavalin A: circularly permuted amino acid sequences. Proc. Natl. Acad. Sci. 76: 3218–3222. Ferrige AG, Seddon MJ, Green BN, Jarvis SA, Skilling J, Staunton J. 1992. Disentangling electrospray spectra with maximum entropy. Rapid Commun. Mass Spectrom. 6: 707–711. Finney DJ. 1971. Probit analysis 3rd edn, vol. 18. University Press: Cambridge, UK; 37–77. Gadelha CAA, Moreno FBMB, Santi-Gadelha T, Cajazeiras JB, Rocha BAM, Assreuy AMS, Mota MRL, Pinto NV, Meireles AVP, Borges JC, Freitas BT, Canduri F, Souza EP, Delatorre P, Criddle DN, Azevedo WF, Cavada BS. 2005. Native crystal structure of a nitric oxide releasing lectin from the seeds of Canavalia maritima. J. Struct. Biol. 152: 185–194. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and analysis tools on the
Revised: 05 November 2013,
Accepted: 08 November 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2340
Purification, characterization and partial sequence of a pro-inflammatory lectin from seeds of Canavalia oxyphylla Standl. & L. O. Williams Mayara Q. Santiagoa, Cintia C. F. Leitãoa, Francisco N. Pereira-Juniora, Vanir R. Pinto-Juniora, Vinicius J. S. Osternea, Claudia F. Lossioa, João B. Cajazeirasa, Helton C. Silvaa, Francisco V. S. Arrudac, Livia P. Pereirad, Ana M. S. Assreuyd, Kyria S. Nascimentoa, Celso S. Naganob and Benildo S. Cavadaa* 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. Keywords: plant lectin; Canavalia oxyphylla; ESI mass spectrometry; toxic effect; pro-inflammatory
INTRODUCTION
J. Mol. Recognit. 2014; 27: 117–123
a M. Q. Santiago, C. C. F. Leitão, F. N. Pereira-Junior, V. R. Pinto-Junior, V. J. S. Osterne, B. S. Cavada Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza, CE, 60440-970, Brazil b C. S. Nagano Laboratório de Espectrometria de Massa aplicado a Proteínas (LEMAP), Federal University of Ceará, Av. Humberto Monte s/n, Bloco 825, Campus do Pici, Fortaleza, CE, 60440-970, Brazil c F. V. S. Arruda Laboratório Integrado de Biomoléculas (LIBS), Integrated Laboratory of Biomolecules (LIBS), Department of Pathology and Legal Medicine, Federal University of Ceará, Rua Monsenhor Furtado, S/N—Rodolfo Teófilo, Fortaleza, CE, 60441-750, Brazil d L. P. Pereira, A. M. S. Assreuy Laboratório de Fisiofarmacologia da Inflamação (LAFFIM), Institute of Biomedical Sciences, State University of Ceara, Av Paranjana 1700, 60740-000, Fortaleza, Ceará, Brazil
Copyright © 2014 John Wiley & Sons, Ltd.
117
Lectins are defined as a structurally heterogeneous group of proteins or glycoproteins that possess at least one noncatalytic domain that binds reversibly to a specific monosaccharide or oligosaccharide (Peumans and Van Damme, 1995a, 1995b). Lectins have been studied extensively in recent years based on their overall utility as molecular tools involving (i) cell surface composition, growth, and differentiation; (ii) pathological mechanisms; and (iii) the isolation and characterization of glycoconjugates (Sharon and Lis, 2004; Sharon, 2007). Legume lectins constitute the best-studied group of plant lectins, and hundreds of them have been isolated and characterized in relation to their chemical, physicochemical, structural, and biological properties (Van Damme et al., 1998; Peumans and Van Damme, 1995a, 1995b). Specifically, legume lectins are normally composed of two or four monomers, presenting a molecular mass of about 25–30 kDa, with each monomer presenting a unique, highly conserved carbohydrate binding site, as well as conserved metal binding sites for divalent cations (calcium and
* Correspondence to: Benildo S. Cavada, Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza, CE 60440-970, Brazil. E-mail: [email protected]
M. Q. SANTIAGO ET AL. manganese). The monomers are associated by noncovalent interactions (Brinda et al., 2004). Lectins of the Diocleinae subtribe are highly homologous with regard to carbohydrate binding specificities and biochemical and physicochemical properties, but they differ in biological activity (Barral-Netto et al., 1992; Assreuy et al., 2009). In general, Diocleinae lectins elicit anti-inflammatory or pro-inflammatory responses, depending on the administration route used, being inhibitory when administered systemically, but stimulatory when applied locally. ConBr, a Diocleinae lectin extracted from Canavalia brasiliensis, is an exception, producing only proinflammatory responses (Assreuy et al., 1997). Minor differences in the ratios of dimeric and tetrameric forms in the lectins, together with differences in the relative orientations of the carbohydrate-binding sites in the quaternary structures, have been hypothesized to contribute to the differences in biological activities exhibited by Diocleinae lectins (Cavada et al., 1994). The genus Canavalia comprises 51 species of leguminous vines, which are widespread in Brazil, Guiana, and the Atlantic coastal regions of tropical Central America and Mexico (Lackey, 1981). In the present study, we isolated a glucose/mannosebinding lectin [Canavalia oxyphylla Lectin (CoxyL)] from the seeds of Canavalia oxyphylla Standl. & L. O. Williams, a woody vine of the Diocleinae subtribe. The purified lectin was physicochemically characterized, in particular, its toxic effect on Artemia sp. nauplii, the brine shrimp, and its pro-inflammatory activity.
Hemagglutination activity and sugar specificity The hemagglutination assay was performed in microtiter plates by serial dilution using 3% native rabbit erythrocytes treated with the proteolytic enzymes trypsin and papain (Ainouz et al., 1992). The carbohydrate binding specificity of CoxyL was determined by the ability of different sugars and glycoproteins to inhibit erythrocyte agglutination (Ramos et al., 1996). Serial dilution was performed on sugars/glycoproteins (100 mM/1 mg/ml initial concentrations, respectively, and CoxyL was subsequently added to each dilution at a concentration of 4 hemagglutinating unit [HU]). HU was expressed as the value of the highest dilution giving positive hemagglutination per milliliter of sample, and inhibition values were expressed by minimum inhibitory concentration. Effects of pH, temperature, and divalent ions on lectin-induced hemagglutination
MATERIAL AND METHODS Purification of Canavalia oxyphylla lectin The seeds obtained were peeled and ground in a coffee grinder (CadenceTM MDR301 Monovolt). After seed flour obtained were stored in an oven at a temperature of 37°C, dry flour was obtained. Soluble proteins were extracted in 150 mM NaCl [1:10 (w: v)] under constant stirring for 4 h at room temperature. Subsequently, the extract was centrifuged at 10 000 × g at 4°C for 20 min, and the supernatant was filtered on filter paper porosity 6 μm (WhatmanTM). The resulting supernatant was applied to Sephadex® G-50 affinity column (6.5 × 1.5 cm) previously equilibrated with 150 mM NaCl. Unbound material (P1) was washed with the same solution, and lectin was eluted with 100 mM D-glucose in the equilibrium solution. To estimate protein concentration, the eluted fractions were monitored by absorbance at 280 nm and by Bradford (1976), using bovine serum albumin as standard. The eluate was collected, dialyzed against distilled water, and lyophilized. The homogeneity of the sample was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the pure protein was used for characterization tests. Electrophoretic analysis
118
The purity and apparent molecular weight of the lectin were determined by PAGE in the presence of SDS (SDS-PAGE). The experiment was performed in a discontinuous buffer system as described by Laemmli (1970) using 15% total concentration polyacrylamide as the separating gel and 4% total concentration polyacrylamide as the stacking gel. The procedure was carried out using a Mini Protean II apparatus (BioRad; Milan; Italy) under constant current of 25 mA for 60 min. At the end of electrophoresis, the separated protein bands were stained with Coomassie
wileyonlinelibrary.com/journal/jmr
Brilliant Blue R-250. The following molecular markers were used: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). The presence of disulfide bonds was tested by addition of 2% β-mercaptoethanol in the sample prior to the electrophoretic run. The presence of covalently bound carbohydrate was evaluated by staining with periodic acid-Schiff’s reagent after SDS-PAGE (Zacharius et al., 1969).
The pH stability of CoxyL was determined by dissolving the lectin in different buffers, all containing 150 mM NaCl, and measuring the hemagglutinating activity of the solutions. The buffers used in this test were 100 mM sodium citrate for pH 4.0 and 6.0, 100 mM sodium acetate for pH 5.0, 100 mM sodium phosphate for pH 7.0, 100 mM Tris–HCl for pH 8.0, and 100 mM GlycineNaOH for pH 9.0 and 10.0. To investigate the thermal stability, CoxyL (0.125 mg/ml) was prepared in 150 mM NaCl and incubated at various temperatures (40, 50, 60, 70, 80, 90, and 100°C) for 60 min. After incubation, the solutions were cooled to room temperature, and their hemagglutinating activity was determined. For analysis of the influence of divalent cations on the lectin hemagglutinating activity, CoxyL was dialyzed against 25 mM ethylenediaminetetraacetic acid (EDTA) containing 150 mM NaCl for 24 h at 4°C, followed by an additional dialysis against 150 mM NaCl. HU of treated and untreated samples were compared. Molecular mass determination The average molecular mass of C. oxyphylla lectin was determined by electrospray ionization mass spectrometry using a hybrid quadrupole/ion mobility separator/orthogonal acceleration time-of-flight mass spectrometer (SYNAPT HDMS System, Waters Corp., Milford, MA, USA). C. oxyphylla lectin ( ρmol/μl in 50% acetonitrile with 0.1% formic acid) was infused into the system at 0.5 μl/min. The capillary and cone voltages were set at 3.5 kV and 50 V, respectively. The source temperature was maintained at 80°C, and nitrogen was used as a drying gas (flow rate of 100 l/h). The instrument was calibrated with fragments of the double protonated ion of [Glu1]-fibrinopeptide B (SigmaAldrich, St. Louis, MO, USA) (m/z 785.84). Data acquisition was performed at m/z in the range of 800–3000, using MASS LYNX 4.0 software (Waters), and the spectra of multiply charged
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL ions were deconvoluted using maximum entropy techniques (Ferrige et al., 1992).
of research animals (National Institute of Health guidelines) and approved by the Institutional Animal Care and Use Committee of the State University of Ceará (UECE–N° 0559924–4).
Proteolytic cleavages Protein digestion was performed as previously described by Shevchenko et al. (2006). To accomplish this, C. oxyphylla lectin was submitted to SDS-PAGE, and the bands were excised and bleached with 50 mM ammonium bicarbonate in 50% acetonitrile. Bands were dehydrated in 100% acetonitrile and dried in a Speedvac (LabConco, Kansas City, MO, USA). For proteolytic cleavage, gels were rehydrated in an enzyme solution of 50 mM ammonium bicarbonate containing trypsin (Promega, Madison, WI, USA) at 37°C overnight. The obtained peptides were extracted in a solution of 50% acetonitrile with 5% formic acid and concentrated in Speedvac.
Effect of CoxyL on rat paw edema Paw edema was measured immediately before (zero time) intraplantar subcutaneous (s.c.) injection of CoxyL (0.01, 0.1, and 1 mg/Kg) and thereafter (30–600 min) using a hydroplethysmometer (PanLab, Barcelona, Spain). Results were expressed as the increase in paw volume (ml) calculated by subtracting the basal volume measured at zero time or area under curve (AUC) (arbitrary units) (Landucci et al., 1995). Data are presented as mean ± S.E.M. The statistical differences, set at p < 0.05, were analyzed using analysis of variance followed by Bonferroni’s test.
Tandem mass spectrometry analysis The peptides obtained in the different proteolytic cleavages were separated on a BEH300 C18 column (100 × 100 mm) using a nanoAcquity™ system and eluted with acetonitrile gradient (10–85%), containing 0.1% formic acid at 600 μl/min. The liquid chromatography system was connected to a nanoelectrospray mass spectrometer source (SYNAPT HDMS System, Waters Corp., Milford, USA). The mass spectrometer was operated in positive mode, using a source temperature of 90°C and capillary voltage of 3.5 kV. The lock mass used in acquisition was m/z 785.84 ions of the [Glu1] fibrinopeptide B. The LC-MS/MS experiment was performed according to the data dependent acquisition function, selecting MS/MS doubly or triply charged precursor ions, which were fragmented by collision-induced dissociation (CID) using argon as collision gas and ramp collision energy that varied according to the charge state of the selected precursor ion. The CID spectra were interpreted manually using the Peptide Sequencing tool from MASS LYNX 4.0 software (Waters). The C. oxyphylla lectin peptide sequences were compared with all non-redundant proteins deposited in the National Center of Biotechnology Information using BLAST (Altschul et al., 1997), and the proteins with the best e-value were selected to sequence alignments at ClustalW (Gasteiger et al., 2005).
RESULTS AND DISCUSSION Purification of CoxyL and hemagglutination assays The crude extract of C. oxyphylla seeds showed strong agglutination activity in native rabbit erythrocytes or treated with trypsin and papain. The inhibition assay of hemagglutinating activity with carbohydrates showed that the lectin has specificity for D-glucose, D-mannose, and α-methyl-D-mannoside. Glycans present in ovalbumin and fetuin were able to inhibit the hemagglutinating activity of CoxyL, perhaps by the presence of mannose in its composition (Table 1). The specificity profile of CoxyL is similar to that of other lectins in the Canavalia genus. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column, in which the lectin was quantitatively retained in the gel and eluted with 100 mM D-glucose (Figure 1A). The soluble protein content and the specific activity of the crude lectin extract were 10.8 mg/ml and 95 HU/mg protein, respectively. For the purified lectin, the values were 0.82 mg/ml and 2498 HU/mg protein, respectively. The specific activity increased by 26.3-fold in the pure lectin (Table 2).
Artemia lethality test CoxyL (200 μg/ml) was homogenized with artificial sea water and assayed in triplicate on Linbro plates 24 wells, containing 10 specimes of Artemia sp. nauplii per well with a final volume of 2 ml. Lectin solution was added to the wells to reach concentrations of 12.5, 25, 50, and 100 μg/ml compared with controls containing sea water only. To block the activity of the lectin, CoxyL (12.5, 25, 50, and 100 μg/ml) was incubated in artificial sea water containing 100 mM of mannose for 1 h at 37°C. After 24 h, the percentage of deaths and LC50 values were obtained using the simple Microsoft Excel 2013 and calculated by probit analysis as described by Finney (1971). Pro-inflammatory activity Animals
J. Mol. Recognit. 2014; 27: 117–123
Carbohydrates and glucoproteins D-glucose D-mannose D-galactose N-acetyl-D-glucosamine α-methyl-D-mannoside α-methyl-D-galactoside α-lactose β-lactose Ovalbumin Fetuin
MIC 50 mM 25 mM NI NI 12.5 mM NI NI NI 0.25 mg/ml 0.125 mg/ml
MIC, minimum inhibitory concentration; NI, sugar not inhibitory until a concentration of 100 mM is reached.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
119
Male Wistar rats (150–250 g), n = 6–7 animals per group, were maintained in a controlled 12/12 h light/dark cycle, at 25°C with free access to food and water. Experiments were conducted in accordance with current guiding principles for the care and use
Table 1. Inhibitory effect of saccharides and glycoproteins on the hemagglutinating activity of Canavalia oxyphylla lectin
M. Q. SANTIAGO ET AL. Effects of pH, temperature, and divalent ions on lectin-induced hemagglutination The effect of temperature on CoxyL activity is shown in Figure 2A. The lectin was shown to be thermostable, exhibiting full activity up to 60°C, with a considerable loss in activity occurring at 70°C (loss of 87.5%), and no hemagglutinating activity was detected at 90°C. Examination of CoxyL activity toward different pH values showed that the lectin was pH-sensitive. CoxyL exhibited maximum activity at pH 7.0, indicating that the lectin is more stable in this condition, while in acidic pH (4.0–6.0), the lectin showed low stability with only 25% of its hemagglutinating activity remaining. In basic pH (8.0–9.0), the lectin lost 50% of its activity in pH 8.0, and a further decrease in pH beyond 9.0 caused a 75% loss in hemagglutination activity in pH 9.0 (Figure 2B). The thermostability and pH stability of CoxyL are similar in related Diocleinae lectins (Moreira et al., 1995; Correia et al., 2011). After treating the lectin with EDTA, the hemagglutination activity of CoxyL remained unchanged. These data suggest that CoxyL, in contrast to various other leguminous lectins (Cavada et al., 2001; Loris et al., 1998), does not need divalent cations for its activity, as they are most probably tightly bound to the protein. Although this property is especially rare in the case of lectins from the Diocleinae subtribe, the hemagglutinating activity of other Diocleinae lectins, such as those from Canavalia cathartica, was also unaffected by EDTA (Suseelan et al., 2007).
Mass spectrometry analysis Figure 1. CoxyL purification and molecular mass estimation. (A) Elution profile of Sephadex® G-50 affinity chromatography. Approximately 10 ml of the crude extract was applied to the Sephadex® G-50 column (6.5 × 1.5 cm) that was equilibrated with 150 mM NaCl. The lectin was eluted with 100 mM D-glucose at a flow rate of 1 ml/min. Fractions (approximately 1.5 ml) were collected and monitored for protein content by measuring the absorbance at 280 nm. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lane 1: molecular mass markers (phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; trypsin inhibitor and a-lactalbumin, 14.4 kDa); lane 2: Crude extract of C. oxyphylla (15 μg); lane 3: CoxyL (15 μg).
Electrophoretic analysis and presence of covalently bound carbohydrate The electrophoretic profile obtained with SDS-PAGE of the affinity-purified CoxyL, both in the presence and absence of β-mercaptoethanol, revealed a major band of 30 kDa (α-chain) (Figure 1B). The β-fragments and γ-fragments were not observed on SDS-PAGE a typical ConA-like lectin, perhaps because of low concentration of these fragments in the purified sample.
Electrospray ionization mass spectrometry confirmed that CoxyL consists of a combination of chains weighing 25 696 ± 2 Da (α-chain), 12 980 ± 2 Da (β-chain), and 12 733 ± 2 Da (γ-chain) (data not shown), suggesting that C. oxyphylla lectin is a typical ConA-like lectin subject to the posttranslational processing of circular permutation described by Carrington et al. (1985). Thus, CoxyL should be expressed as a pre-pro-protein (Ntermsignal peptide + γ-chain + linker peptide + β-chain + Ctermsignal peptide) cleaved into a γ product and a β product. The active protein is a final fused product (α-chain) with the two fragments in inverse order and having no signal or linker peptides (Cunningham et al., 1979; Carrington et al., 1985; Chrispeels et al., 1986). The β-fragment and γ-fragment observed on SDS-PAGE are unlinked products of this process, while the α-chain is the mature protein. CoxyL showed no staining by periodic acid-Schiff, suggesting that it is not a glycoprotein. The partial amino acid sequence of CoxyL was determined by tandem mass spectrometry after proteolysis of the protein band excised from an SDS-PAGE gel. Six peptides were sequenced, resulting in a total of 107 amino acid residues (Table 3). In
Table 2. Purification of lectin from Canavalia oxyphylla seeds Fraction
a
Total protein (mg/ml)
Crude extract PII (Sephadex® G-50)
10.8 0.82
b
Total HU
c
Specific activity (HU/ml)
10
2 211
95 2498
Purification (fold) 1 26.3
a
Protein content. Hemagglutinating activity expressed in hemagglutinating units (HU). c Specific activity calculated as the ratio between hemagglutinating activity and protein content. b
120
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL wilsonii (DwL) (SwissProt accession code: P86624), and Cratylia floribunda (CFL). In relation to ConA, the partial amino acid sequence of CoxyL showed differences in seven positions (ConA/CoxyL): A50V and D59G in T3; S96T in T1; H121A, M129T, and S134T in T4; and E155Q in T5. Legume lectins of the Diocleinae subtribe present similar carbohydrate-binding specificities and reveal considerable homology in amino acid sequence and tertiary structure (Calvete et al., 1999; Cavada et al., 2001). Nevertheless, Diocleinae lectins may differ substantially in biological activities (Barral-Netto et al., 1992; Gadelha et al., 2005; Assreuy et al., 2009). These differences can be associated with the position of amino acids in the carbohydrate-binding site or residues located in key positions of the quaternary structure (Calvete et al., 1999; Wah et al., 2001; Brinda et al., 2004; Nagano et al., 2008). Lethality test
Figure 2. Physicochemical properties of the CoxyL. Hemagglutinating activity of the PFL after different treatments by temperatures (A) and pH measures (B).
comparison with average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. Figure 3 shows the CID spectra of peptide T3, in which sequence-specific y-ions were used for sequence determination. Comparisons with other sequences from the National Center of Biotechnology Information databank revealed that the CoxyL peptides were similar to other Diocleinae lectins, such as Canavalia ensiformis (ConA) (SwissProt accession code: P02866), Canavalia brasiliensis (ConBr) (SwissProt accession code: P55915), Canavalia gladiata (CGL) (PDB accession code: 2EF6), Canavalia maritima (CML) (PBD accession code: 2P34), Dioclea guianensis (Dgui) (SwissProt accession code: P81637), Dioclea grandiflora (DGL) (SwissProt accession code: A9J251), Dioclea
CoxyL has toxic activity against brine shrimp (Artemia sp. nauplii), exhibiting LC50 value of 44.31 μg/ml. This effect proved to be dose dependent and was observed in a concentration range from 12.5 to 100 μg/ml (Figure 4A). The Artemia lethality test (Persoone, 1980; Sorgeloos et al., 1978) has been successfully used to determine the toxicity of biological molecules that have a variety of pharmacological activities, including anticancer agents, antivirals, insecticides, pesticides, and anti-HIV compounds (Carballo et al., 2002; Pervin et al., 2006; Ho et al., 2007). Previous work showed that other lectins, such as ConA-like, also have toxic effect (Santos et al., 2010), with LC50 between 2.52 and 15.5 μg/ml. In comparison to similar lectins, CoxyL exhibited low toxicity against Artemia. The lethality of animals abolished when the lectin was incubated with D-mannose (Figure 4B), showing that the carbohydrate recognition domain is responsible for the toxic activity against Artemia sp. Canavalia oxyphylla lectin presents edematogenic effect CoxyL induced an increase in paw edema at 0.01 mg/kg (116.7 ± 22.46 AUC), 0.1 mg/kg (138.6 ± 26.00 AUC), and 1 mg/kg (349.5 ± 28.39 AUC) compared with the saline group (18.3 ± 5.73 AUC). At the highest dose (1 mg/kg), local injection of CoxyL evoked significant paw edema in the initial 30 min, that lasted until 540 min (0.1 ± 0.03 ml), with maximal activity at 120 min (1.1 ± 0.05 ml) compared with saline (0.08 ± 0.03 ml) (Figure 5). The edematogenic effect elicited by CoxyL was characteristic of acute inflammatory responses. Similar responses had been
Table 3. Amino acid peptide sequences of CoxyL obtained using tandem mass spectrometry and their respective molecular masses Peptide* T1 T2 T3 T4 T5 T6
Experimental mass (Da)
Theoretical mass (Da)
Sequence**
1108.6128 1512.7460 1385.7667 2175.0127 2545.0549 3293.6091
1108.5044 1512.7244 1385.7065 2175.9165 2545.9644 3293.5564
VGXSATTGXYK ETNTXXSWS FTSK VGTVHXXYNSVGK SNSTAETNAXHFTFNQFTK DXXXQGDATTGTDGNLQXTR XSAVVSYPNADSATVSYDVDXDNVXPEWVR
J. Mol. Recognit. 2014; 27: 117–123
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
121
*Peptide obtained from cleavage with trypsin (T). **In peptide sequence, X represents residues of leucine or isoleucine, which cannot be distinguished by mass.
M. Q. SANTIAGO ET AL.
Figure 3. Peptide from CoxyL. Collision-induced dissociation of doubly charged ion T2 at m/z 694.39 with sequence-specific y-ion series interpreted as VGTVHXXYNSVGK.
Figure 4. Toxicity to the Artemia sp. nauplii assays. (A) Toxic effect of CoxyL at different concentrations (12.5, 25, 50, or 100 mg/ml) on Artemia nauplii; (B) toxic effect of CoxyL (100 mg/ml), CoxyL (100 mg/ml) previously incubated with 100 mM mannose. Mean_SEM (n = 5). *p < 0.05 compared with control.
122
previously shown for the lectin isolated from Canavalia maritma seeds, in which the edematogenic effect lasted 300 min, while the edematogenic effect induced by ConBr and CGL was more lasting, about 32 h, compared with CoxyL (Assreuy et al., 2009). These data are in line with other reports that highlights the relation structure and biological activities of these homologous lectins (Barral-Netto et al., 1992; Calvete et al., 1999; Gadelha et al., 2005; Cavada et al., 2011).
wileyonlinelibrary.com/journal/jmr
Figure 5. Edematogenic effect of the lectin Canavalia oxyphylla lectin. (A) Paw edema (ml) induced by C. oxyphylla. Animals received CoxyL (0.01; 0.1, and 1 mg/Kg, s.c.) or saline (0.01 ml per paw, s.c.). Edema was measured immediately before (zero time) and thereafter (30–600 min) using a hydroplethysmometery and expressed as (A) variation in paw volume displacement (ml) or (B) area under curve between different times and time zero. Mean ± E.P.M. (n = 6). Analysis of variance and Bonferroni’s test. *p < 0.05 compared with saline.
CONCLUSION CoxyL showed a combination of chains weighing 25 696 ± 2 Da (α-chain), 12 980 ±2 Da (β-chain), and 12 733 ±2 Da (γ-chain).
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 117–123
PURIFICATION, CHARACTERIZATION OF A PRO-INFLAMMATORY NEW LECTIN COXYL It was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). CoxyL demonstrated toxicity against Artemia sp. nauplii with dose-dependent effect, and it showed pro-inflammatory activity in rats by inducing an increase in paw edema. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.
Acknowledgements This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP). A. M. S. A., K. S. N., C. S. N., and B. S. C are senior investigators of CNPq, and David Martin helped with the English editing of the manuscript.
REFERENCES
J. Mol. Recognit. 2014; 27: 117–123
ExPASy server. In The proteomics protocols handbook, Walker JM (ed). Humana Press: Totowa NJ USA; 571–607. Ho JC, Chen CM, Row LC. 2007. Oleanane-type triterpenes from the flowers, pith, leaves, and fruit of Tetrapanax papyriferus. Phytochemistry 68: 631–635. Lackey JA. 1981. Tribe 10. Phaseoleae DC. (1825), In Advances in legume systematics. Part 1, Polhill RM, Raven PH (eds). Royal Botanic Gardens, Kew: UK, 301–327. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680–685. Landucci ECT, Antunes E, Donato JL, Faro RRA, Hyslop S, Marangoni S, Oliveira B, de Nucci G. 1995. Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2. Brit. J. Pharm. 114: 578–583. Loris R, Hamelryck T, Bouckaert J, Wyns L. 1998. Legume lectin structure. Biochim. Biophys. Acta 1383: 9–36. Moreira RA, Cordeiro EF, Cavada BS, Nunes EP, Fernandes AG, Oliveira JTA. 1995. Lectins and the chemotaxonomy of the genus Diocleinae (Leguminosae-Phaseoleae). R. Bras. Fis. Veg. 7(1): 7–14. Nagano CS, Calvete JJ, Barettino D, Pe´rez A, Cavada BS, Sanz L. 2008. Insights in to the structural basis of the pH-dependent dimertetramer Equilibrium through crystallographic analysis of recombinant Diocleinae lectins. Biochem. J. 409: 417–428. Persoone G. 1980. Proceeding of the international symposium on brine shrimp Artemia salina. University Press: Belgim; 1–3. Pervin F, Hossain MM, Khatun S, Siddique SP, Salam KA, Karim MR, Absar N. 2006. Comparative citotoxicity study of six bioactive lectins purified from pondweed (Potamogeton nodosus Poir) rootstock on Brine Shrimp. J. Med. Sci. 6: 999–1002. Peumans WJ, Van Damme EJM. 1995a. Lectins as plant defense proteins. Plant Physiol. 109: 347–352. Peumans WJ, Van Damme EJM. 1995b. The role of lectins in plant defense. Histochem. J. 27: 253–271. Ramos MV, Moreira RA, Cavada BS, Oliveira JTA, Rouge P. 1996. Interaction of lectins from the sub tribe Diocleinae with specific ligands. R. Bras. Fisiol. Veg. 8: 193–199. Santos AF, Cavada BS, Rocha BAM, Nascimento KS, Sant’Ana AEG. 2010. Toxicity of some glucose/mannose-binding lectins to Biomphalaria glabrata and Artemia salina. Bioresour. Technol. 101: 794–798. Sharon N. 2007. Lectins: carbohydrate-specific reagents and biological recognition molecules. J. Biol. Chem. 282: 2753–2764. Sharon N, Lis H. 2004. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14: 53–62. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. 2006. In gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1: 2856–2860. Sorgeloos P, Van Der Wielen CR, Persoone G. 1978. The use of artemia nauplii for toxicity tests—a critical analysis. Ecotoxicol. Environ. Saf. 2: 249–255. Suseelan KN, Bhagwath A, Pandey R, Gopalakrishna T. 2007. Characterization of Con C a lectin from Canavalia cathartica Thouars seeds. Food Chem. 104(2): 528–535. Van Damme EJM, Peumans WJ, Barre A, Rougé P. 1998. A novel family of lectins evolutionarily related to class V Chitinases: an example of neofunctionalization in legumes. Crit. Rev. Plant Sci. 17: 575–692. Wah DA, Romero A, Gallego del Sol F, Cavada BS, Ramos MV, Grangeiro TB, Sampaio AH, Calvete JJ. 2001. Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association. J. Mol. Biol. 310: 885–894. Zacharius RM, Zell TE, Morrison JH, Woodlock JJ. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30(1):148–152.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
123
Ainouz IL, Sampaio AH, Benevides NMB, Freitas ALP, Costa FHF, Carvalho MR, Joventino FP. 1992. Agglutination of enzyme treated erythrocytes by Brazilian marine algae extracts. Botânica Marinha 35: 447–479. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17): 3389–402. Assreuy AMS, Shibuya MD, Martins GJ, Souza MLP, Cavada BS, Moreira RA, Oliveira JTA, Ribeiro RA, Flores CA. 1997. Anti-inflammatory effect of glucose-mannose binding lectins isolated from Brazilian beans. Mediat. Inflamm. 6: 201. Assreuy AMS, Fontenele SR, Pires AF, Fernandes DC, Rodrigues NV, Bezerra EH, Moura TR, Nascimento KS, Cavada BS. 2009. Vasodilator effects of Diocleinae lectins from the Canavalia genus. N-S Arch. Pharmakol. 380: 509. Barral-Netto M, Santos SB, Barral A, Moreira LIM, Santos CF, Moreira RA, Oliveira JTA, Cavada BS. 1992. Human lymphocyte stimulation by legume lectins from the Diocleae tribe. Immunol. Invest. 21: 297. Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein-dye binding. Anal. Biochem. 72: 248–254. Brinda KV, Mitra N, Surolia A, Vishveshwara S. 2004. Determinants of quaternary association in legume lectins, Protein Sci. 13: 1735–1749. Calvete JJ, Thole HH, Raida M, Urbanke C, Romero A, Grangeiro TB, Ramos MV, Rocha IMA, Guimarães FN, Cavada BS. 1999. Molecular characterization and crystallization of Diocleinae lectins. Biochim. Biophys. Acta 1430: 367–375. Carballo JL, Hernández-Inda ZL, Pérez P, García-Grávalos MD. 2002. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnol. 2: 17–21. Carrington DM, Auffret A, Hanke DE. 1985. Polypeptide ligation occurs during post-translational modification of Concanavalin A. Nature 313: 64–67. Cavada BS, Grangeiro TB, Ramos MV, Crisostomo CV, Silva LM, Moreira RA, Oliveira JTA. 1994. Lectin from Dioclea guianensis var. lasiophylla Duke seeds mobilization during germination and seedlings growth in the dark. Rev. Bras. Fisiol. Veg. 6: 21–25. Cavada BS, Barbosa T, Arruda S, Grangeiro TB, Barral Netto M. 2001. Revisiting proteus: do minor changes in lectin structure matter in biological activity? Lessons from and potential biotechnological uses of the Diocleinae subtribe lectins. Curr. Protein Pept. Sci. 2: 123–135. Chrispeels MJ, Hartl PM, Sturm A, Faye L. 1986. Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. J. Biol. Chem. 261: 10021–10024. Correia JL, do Nascimento AS, Cajazeiras JB, Gondim AC, Pereira RI, de Sousa BL, da Silva AL, Garcia W, Teixeira EH, do Nascimento KS, da Rocha BA, Nagano CS, Sampaio AH, Cavada BS. 2011. Molecular characterization and tandem mass spectrometry of the lectin extracted from the seeds of Dioclea sclerocarpa Ducke. Molecules 16: 9077–9089. Cunningham BA, Hemperly JJ, Hopp TP, Edelman GM. 1979. Favin versus concanavalin A: circularly permuted amino acid sequences. Proc. Natl. Acad. Sci. 76: 3218–3222. Ferrige AG, Seddon MJ, Green BN, Jarvis SA, Skilling J, Staunton J. 1992. Disentangling electrospray spectra with maximum entropy. Rapid Commun. Mass Spectrom. 6: 707–711. Finney DJ. 1971. Probit analysis 3rd edn, vol. 18. University Press: Cambridge, UK; 37–77. Gadelha CAA, Moreno FBMB, Santi-Gadelha T, Cajazeiras JB, Rocha BAM, Assreuy AMS, Mota MRL, Pinto NV, Meireles AVP, Borges JC, Freitas BT, Canduri F, Souza EP, Delatorre P, Criddle DN, Azevedo WF, Cavada BS. 2005. Native crystal structure of a nitric oxide releasing lectin from the seeds of Canavalia maritima. J. Struct. Biol. 152: 185–194. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and analysis tools on the
