Research Article Received: 28 July 2014,
Revised: 3 October 2014,
Accepted: 4 October 2014,
Published online in Wiley Online Library: 23 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2441
The galactose-binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines Deysen Kerlla Fernandes Bezerra Girãoa, Benildo Sousa Cavadab, Alana de Freitas Piresc, Timna Varela Martinsc, Álvaro Xavier Francoa, Cecília Mendes Moraisa, Kyria Santiago do Nascimentob, Plinio Delatorred, Helton Colares da Silvab, Celso Shiniti Naganoe, Ana Maria Sampaio Assreuyc and Pedro Marcos Gomes Soaresa* 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. Keywords: Bauhinia bauhinioides; galactose-binding lectin; anti-inflammatory effect; neutrophil; TNF-α
INTRODUCTION
to their effects on inflammatory processes. The anti-inflammatory effect of plant lectins has been shown to be primarily because of
J. Mol. Recognit. 2015; 28: 285–292
* Correspondence to: P. Marcos Gomes Soares, Departamento de Morfologia, Universidade Federal do Ceará, Rua Delmiro de Farias s/n, 60430-170, Fortaleza, Ceará, Brazil. E-mail: [email protected] a D. K. F. B. Girão, Á. X. Franco, C. M. Morais, P. M. G. Soares Departamento de Morfologia, Universidade Federal do Ceará, Rua Delmiro de Farias s/n, 60430-170, Fortaleza, Ceará, Brazil b B. S. Cavada, K. Santiago do Nascimento, H. C. Silva Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 907, Box 6043, 60440-970, Fortaleza, Ceará, Brazil c A. Pires, T. V. Martins, A. M. S. Assreuy Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, Av. Paranjana 1700, 60740-000, Fortaleza, Ceará, Brazil d P. Delatorre Departamento de Biologia Molecular, Universidade Federal da Paraíba, Cidade Universitária, 58059-900, João Pessoa, Brazil e C. S. Nagano Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 827, Fortaleza, Brazil
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Legumes in the genus Bauhinia (Caesalpinoideae) are widely distributed in most of the tropical regions worldwide including Africa, Asia and South America (Cechinel Filho, 2009). Many biological activities have been identified in compounds from Bauhinia bauhinioides (BBL), being one of the most studied antithrombotic and antitumor effects resulting from the action of kallikrein isolated from this bean (Brito et al., 2014; Bilgin et al., 2014). Another important substance extracted from BBL is a sugar-binding protein, denominated lectin. The property of specifically and reversibly binding to carbohydrates or precipitate glycoconjugates is a feature that enables lectins to interact with a variety of mammalian cells, modulating several biological events, including the inflammatory process (Sharon and Lis, 2004; Souza et al., 2013). In fact, the lectin domain of selectins plays a pivotal role in leukocyte arresting and rolling through reversible binding to complex carbohydrates (Kansas, 1996; Malik and Lo, 1996). However, the mechanism by which plant lectins interfere with the inflammatory process has not yet been elucidated. Despite differences in carbohydrate-binding specificity and quaternary structures, legume lectins present high sequential identity (Cavada et al., 2001) but differ in potency and efficacy with respect
D. K. F. B. GIRÃO ET AL. Table 1. Amino acid sequences and molecular masses of Bauhinia bauhinioides peptides obtained by tandem mass spectrometry Peptidea T1 T2 T2′ T3 T4 T5 T6 T7 T8
Experimental mass (Da)
Theoretical mass (Da)
Sequence
1313.7383 1436.7736 1464.7684 1870.8809 803.4145 1902.9476 1596.7823 1966.8909 2525.0498
1313.6444 1436.6044 1464.7644 1870.9201 803.4089 1902.7843 1596.7644 1966.8043 2525.1343
AXFXAPPDFPVK HXGXNVNSXESVR HXGXNVNDXESVR AXYVAPXHXWDDTDSR NSXESVR YXXSWSFSSTTTNXQR TDSFYTTFTFVXR DVFSESVATAHVGYDNEK WEDKDVFSESVATAHVGYDNEK
a Peptide obtained from cleavage with trypsin (T). In peptide sequence, X represents leucine or isoleucine residues, which cannot be distinguished by mass.
interference with neutrophil migration via the inhibition of primary cytokines (TNF-α and IL1-β) and interaction with the lectin domain (Assreuy et al., 1997; Assreuy et al., 1999; Mota et al., 2006; Napimoga et al., 2007; Pinto et al., 2013). Most of these studies were performed in rodents via systemic treatment with lectins that displays binding specificity for glucose–mannose (Assreuy et al., 1999; Assreuy et al., 1997; Rocha et al., 2011; Simoes et al., 2012; Pinto et al., 2013; Silva et al., 2013) and N-acetylglucosamine (Alencar et al., 1999; Alencar et al., 2005; Napimoga et al., 2007). However, the anti-inflammatory effect of galactose-binding lectins has only been demonstrated for those isolated from the leguminous Luetzelburgia auriculata (Alencar et al., 2010). The lectin of BBL, which displays binding affinity for galactose, has a molecular mass of 28.310 Da as demonstrated by mass spec-
trometry. BBL lectin exhibits hemagglutinating activity against rabbit erythrocytes and edematogenic effects in rats through carbohydrate site interaction (Silva et al., 2011). Thus, the present study aimed to reveal the partial amino acid sequence of the lectin isolated from the leguminous BBL and to investigate whether this lectin possesses anti-inflammatory properties in acute models of inflammation in rats.
MATERIALS AND METHODS Drugs and reagents Dextran, carrageenan, TNF-α, ammonium bicarbonate, D-galactose, formic acid, o-dianosidine dihydrochloride and [Glu1]-fibrinopeptide B were purchased from Sigma (St. Louis, MO, USA); acetonitrile was
A
B
286
Figure 1. Mass spectra of Bauhinia bauhinioides (BBL) isoform peptide sequences. Collision-induced dissociation of two isoforms of T2 ion. (A) A doubly charged ion at m/z 719.83 with sequence-specific y-ion series interpreted as HXGXNVNSXESVR and (B) a doubly charged ion at m/z 733.39 with sequence-specific y-ion series interpreted as HXGXNVDSXESVR. The letter X in peptide sequence represents leucine or isoleucine residues, which cannot be distinguished by mass.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2015; 28: 285–292
THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides purchased from Tedia (Rio de Janeiro, Brazil); trypsin from Promega (Madison, WI, USA); sterile saline 0.9% was purchased from Fresenius Kabi Brasil (Aquiraz, Ceará, Brazil); 5-5′-dithiolbis2-nitrobenzoic acid and Tris-buffer were purchased from Dinâmica Química Contemporânea Ltda (Diadema, São Paulo, Brazil); 2,2,2-tribromoethanol (Avertin®) was purchased from Chemada Fine Chemicals Company Ltda (Nir Itzhak, D.N. HaNegvev, Israel); and kits for ELISA (TNF-α and IL-1β) were obtained from R&D Systems (Minneapolis, MN, USA). All other drugs and reagents were of analytical grade. Partial amino acid sequence Proteolytic cleavage Bauhinia bauhinioides was submitted to SDS-PAGE (Laemmli, 1970), and the bands were excised, bleached with 50 mM ammonium bicarbonate in 50% acetonitrile, dehydrated in 100% acetonitrile and dried in a Speedvac (LabConco, Kansas City, MO, USA). For proteolytic cleavage, gels were rehydrated in 50 mM ammonium bicarbonate containing trypsin at 37 °C overnight. The obtained peptides were extracted in 50% acetonitrile containing 5% formic acid and concentrated in Speedvac.
Tryptic peptides were separated in BEH300 C18 columns (100 μm × 100 mm) using the nanoAcquity™ (Waters Technologies of Brazil Ltda, Barueri, São Paulo, Brazil) system and eluted at 600 μL/min with acetonitrile gradient (10–85%) containing 0.1% formic acid. The liquid chromatography system was connected to a nanoelectrospray mass spectrometer source (SYNAPT HDMS system, Waters Corp., Milford, MA, USA). The mass spectrometer was operated in positive mode using a source temperature of 90 °C and capillary voltage of 3.5 kV. The instrument was calibrated with fragments of the double protonated ion [Glu1]-fibrinopeptide B (m/z 785.84), and the Lock mass used during the acquisition was the intact ion. The LC–MS/MS procedure was performed according to the data dependent acquisition function, selecting MS/MS doubly or triply charged precursor ions. Ions were fragmented by collision-induced dissociation using argon as the collision gas and ramp collision energy that varied according to the charge state of the selected precursor ion. Data acquisition was performed at an m/z range of 400–2100 for the MS survey and at an m/z range of 50–3000 for MS/MS. Data were processed with MassLynx 4.0 software (Waters Corp.). The collision-induced dissociation spectra were interpreted manually using the Peptide Sequencing tool in MassLynx 4.0 software. BBL peptide sequences were compared
B
A 1,5
*
0,9
1,2
*
*
#
#
* 0,6
Edema (ml)
Saline Cg (1%) Cg + BBL (0.01mg/kg) Cg + BBL (0.1mg/kg) Cg + BBL (1mg/kg)
1,2
Edema (ml)
Tandem mass spectrometry
Saline Cg (1%) Cg + BBL (1mg/kg) Cg + BBL (1mg/kg) + galactose [0.1M] Cg + galactose [0.1M] Cg + Indo (10mg/kg)
0,9
* 0,6
#
#
a
a
* 0,3
0,3
*
a
0,0
0,0 0
1
2
3
4
0
1
2
Time(h)
3
4
Time (h)
C Saline Cg (1%) Cg +BBL + galactose
Cg + BBL (1 mg/kg) galactose (0.1 M)
AUC (arbitrary units)
4 First phase
Second phase
**
3
*
2
# 1
*
0
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Figure 2. The inhibitory effect of Bauhinia bauhinioides (BBL) on the paw edema induced by carrageenan. Animals were treated intravenously with saline, D-galactose, BBL or BBL (1 mg/kg) with D-galactose (0.1 M) or subcutaneous indomethacin (indo) (10 mg/kg), 30 min before carrageenan (Cg). Edema was measured at 30 min, 1, and 2–4 h after Cg and is expressed as the variation in paw volume (ml) or area under curve (AUC) (arbitrary units) in relation to baseline. (A) Dose response curve, (B) reversal of the effect of BBL by incubation with ligand sugar and (C) AUC first and second phase. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg, **p < 0.05 versus Cg + BBL, “a” p < 0.05 Cg versus Cg + indo.
D. K. F. B. GIRÃO ET AL. with all of the nonredundant proteins deposited in the National Center of Biotechnology Information using BLAST (Altschul et al., 1997), and the proteins with the best e-values were selected for sequence alignments at ClustalW.
administration of the inflammatory agents. Positive control was indomethacin (10 mg/kg, s.c.).
Experimental models of inflammation
Peritonitis was induced by intraperitoneal (i.p.) injection of carrageenan (1 mg) or TNF-α (0.1 ηg) 30 min after the intravenous treatment with BBL (1 mg/kg). Negative controls received sterile saline (i.p.). After 4 h, peritoneal fluid was collected in 10 ml of saline (5 IU heparin) for total and differential leukocyte counts, which are expressed per ml (de Souza and Ferreira, 1985). The peritoneal fluid stimulated with carrageenan was centrifuged (3000 g × 15 min), and the obtained supernatant was assessed for the content of the cytokines TNF-α and IL-1β (pg/dl) by enzyme-linked immunosorbent assay and myeloperoxidase (MPO) activity (U/ml) (Bradley et al., 1982). The effect of BBL (1 mg/kg) was also evaluated for leukocyte rolling and adhesion by intravital microscopy.
Animals Wistar rats (150–200 g) were kept in cages (six in each) in a controlled environment (circadian cycle, 25 °C, with food and water ad libitum). Experimental protocols were previously approved by the Institutional Animal Care and Use Committee of the State University of Ceará (No. 10130208/40) following the recommendations of the Guide for the Care and Use of Laboratory Animals of the US Department of Health and Human Services (NIH publication No. 85-23, revised 1985). Paw edema Paw volume was measured immediately before (time zero) subcutaneous (s.c.) injection of inflammatory stimuli (carrageenan or dextran; 300 μg/paw) into the hind paw of rats and thereafter at selected time intervals (0.5, 1–4 h) by hydroplethysmometry (Panlab LE 7500, Barcelona, Spain). The results are expressed as the variation in paw volume (ml) in relation to baseline. The area under the time-course curve (AUC) was expressed in arbitrary units (Landucci et al., 1995). BBL (0.01, 0.1 e 1 mg/kg) or saline was administered by intravenous route 30 min prior to the
A
Peritonitis
Intravital microscopy Leukocyte adhesion and rolling were evaluated 4 h after the induction of peritonitis with carrageenan. The animals were anesthetized with tribromoethanol (250 mg/kg, i.p.) before exposition of the mesenteric bed for in situ microscopic examination. Leukocytes rolling was defined as white blood cells that move at a velocity significantly slower than that of erythrocytes in a given microvessel. Leukocytes were considered to
B
Total
Mononuclear
Neutrophil
Eosinophil Total
Neutrophil Eosinophil
*
6,000
**
*
**
#
*
10,000
4,000
2,000
12,000
Mononuclear
Leukocytes/mL
Leukocytes/mL
8,000
# 8,000
*
6,000 4,000
#
2,000
#
0
0 galactose [0.1M] BBL (1mg/Kg)
Saline
Saline
TNF-
(0.1 ng) BBL
Cg (1mg/cavity)
D
C 35
Saline
Cg + BBL
Cg
Cg + BBL + galactose
30
*
**
25 20 15 10 5
2.0
#
Leukocyte adhesion/min
Leukocyte rolling/min
40
1.5
Saline Cg
Cg + BBL Cg + BBL + galactose
* **
1.0
0.5
#
288
Figure 3. The inhibitory effect of Bauhinia bauhinioides (BBL) on leukocyte migration. Animals were treated intravenously with saline, D-galactose (0.1 M), BBL (1 mg/kg) or BBL with D-galactose 30 min before inflammatory stimuli. Total and differential cell counts were performed 4 h after the induction of peritonitis with (A) carrageenan (Cg; 1 mg) or (B) TNF-α (0.1 ηg). The number of (C) rolled and (D) adherent leukocytes were evaluated in mesenteric microcirculation 4 h after Cg by intravital microscopy. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg, **p < 0.05 versus Cg + BBL.
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THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides be adherent to the venular endothelium if they remained stationary for more than 30 s. Rolling and adherent cells were counted for 10 min in a 10 μm segment of postcapillary venules (three per animal, diameter 10–18 μm) and expressed as the number of rolled leukocytes/min and adherent cells/100 μm2 (Fortes et al., 1991).
significantly decreased leukocyte rolling by 58% (11.78 ± 0.547) and adhesion by 68% (0.385 ± 0.066 cells/min) (Figure 3(C, D)). The association of BBL with its carbohydrate ligand galactose completely abolished the inhibitory effect of BBL in the leukocyte migration induced by Cg (Figure 3(A,C,D)).
Lectin and carbohydrate interaction assay
Bauhinia bauhinioides inhibits the increase of inflammatory mediators
Bauhinia bauhinioides (1 mg/kg) was incubated with its binding sugar (D-galactose; 0.1 M) for 1 h at 37 °C to allow lectin–sugar interaction before performing the inflammatory protocols. Lectin and sugar were also incubated in separated solutions under the same conditions as controls.
Carrageenan significantly increased MPO activity (0.73 ± 1.72 U/ml), TNF-α (267.0 ± 29.04 ρg/ml) and IL-1β (632.2 ± 34.25 ρg/ml) levels, as determined in the peritoneal exudate 4 h after challenge. BBL significantly inhibited MPO by 84% (0.12 ± 0.92 U/ml), TNF-α by 68% (85.5 ± 16.3 ρg/ml) and IL-1β by 47% (330.6 ± 39.98 ρg/ml) (Figure 4).
Statistical analysis Statistical analyses and the construction of graphs were performed using the software GraphPad Prism® (GraphPad Software, San Diego, CA, USA). Comparisons were determined by analysis of variance, followed by Bonferroni’s test. Values at p < 0.05 were considered significant. Data are expressed as the mean ± SEM (n = 6).
RESULTS Amino acid sequence analysis The partial amino acid sequence of BBL, which was obtained by MS/MS after proteolysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis bands, revealed eight peptides, resulting in a total of 86 amino acid residues (Table 1). Sequence heterogeneity was observed in one peptide D/S (aspartic acid/serine) (T2/T2′), indicating that BBL preparations contain highly homologous isolectins (Figure 1). Bauhinia bauhinioides anti-inflammatory effect
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Figure 4. Bauhinia bauhinioides (BBL) reduced myeloperoxidade (MPO) activity and cytokine (TNF-α and IL-1β) levels in peritoneal fluid. Animals were injected intravenously with saline or BBL 30 min prior to intraperitoneal injection of carrageenan (Cg). (A) MPO, (B) TNF-α and (C) IL-1β levels were determined by enzyme-linked immunosorbent assay. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg.
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Carrageenan induced a progressive and intense paw edema that was initiated during the first hour, reached maximal values between 3 and 4 h and was reduced by BBL at 1 mg/kg by 21% (3 h) and 19% (4 h) (Figure 2(A)). The anti-inflammatory effect of BBL (AUC: 1.30 ± 0.008), observed only in the second phase of carrageenan-induced edema (AUC: 2.02 ± 0.006), was completely blocked by BBL (Figure 2(B)) in association with galactose (AUC: 2.82 ± 0.21). Galactose per se did not induce any effect (Figure 2(C)). Indomethacin, the positive control, inhibited the carrageenan-induced edema in all time after second hour (Figure 2(B)). In contrast, the osmotic edema elicited by dextran (AUC: 1.191 ± 0.115) was not altered by BBL (AUC: 1.10 ± 0.749) (figure not shown). Moreover, BBL (1 mg/kg) also exhibited inhibitory effects in the peritonitis model. BBL inhibited the leukocyte migration induced by carrageenan (6275 ± 712.2) by 72% (1725 ± 121.6) and by TNF-α (10375 ± 11.58) by 28% (7600 ± 942.2) (Figure 3(A,B)). This effect was more pronounced in neutrophils because BBL inhibited 51% (849.5 ± 165.7) of the neutrophil migration induced by carrageenan (1725 ± 121.6) and 64% (1683 ± 277.8) of that induced by TNF-α (4649 ± 907.2 cells/ml) (Figure 3). Intravital microscopy revealed that carrageenan induced significant increases in endothelial leukocyte rolling (28.32 ± 1.619) and adhesion (1.216 ± 0.189) compared with saline. BBL
D. K. F. B. GIRÃO ET AL.
DISCUSSION
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Interest in studying the effects of plant lectins has been increasing because glycoconjugate interactions have been implicated in inflammation pathways. Therefore, these proteins are structurally correlated and differ in the ability of binding to carbohydrates; these proteins are considered potential tools for application in biotechnology or immunotherapies (Rudiger and Gabius, 2001). In the present study, we demonstrated that BBL exhibits homology with other Bauhinia lectins and shares binding specificity for D-galactose and its derivatives (Irimura and Osawa, 1972; Silva et al., 2007; Silva et al., 2011). In fact, the partial amino acid sequence reported here corresponds to approximately 35% of the total BBL sequence. Comparisons with other sequences from the National Center of Biotechnology Information databank revealed that BBL peptides show identity from 35.87% to 39.13% with other Bauhinia lectins, such as Bauhinia purpurea (Swiss-Prot accession code: P16030.2), Bauhinia variegata (GenBank accession code: ABQ45362.1) and Bauhinia ungulata (GenBank accession code: ABD19775.1), respectively. Also, the partial amino acid sequence exhibited sequence identity to other galactose/N-acetyl galactosamine-specific lectins from the Leguminosaeae family with 44.57% for lectin III of Griffonia simplicifolia (SwissProt accession code: P24146.3), 40.22% for Vatairea macrocarpa (SwissProt accession code: P81371), 35.87% for Robinia pseudoacacia (PDB accession code: 1FNZ) and 34.78% for Sophora japonica (SwissProt accession code: P93538). Sequence heterogeneity was observed in one peptide D/S (T2/T2’), indicating that BBL preparations contain highly homologous isolectins. Similarly, the cloning and sequence of the B. variegata lectin gene have suggested that this protein could be encoded by a family of genes, which led to the identification of two isoforms (Pinto et al., 2008). The present study also demonstrated the anti-inflammatory effect of BBL in cellular events of acute experimental inflammation models, which is relevant because the anti-inflammatory effect of galactose-binding lectins has previously only been demonstrated for those isolated from the leguminous L. auriculata (Alencar et al., 2010). Bauhinia bauhinioides inhibited the paw edema induced by carrageenan, an effect that was restricted to the second phase. This edema is a biphasic temporal phenomenon characterized by an intense leukocyte influx that involves a complex network of inflammatory mediators (Di Rosa et al., 1971). The initial phase (0–2 h) is predominantly vascular, triggered by the release of biogenic amines (histamine, bradykinin and serotonin) (Ferreira et al., 1974), whereas the second phase (2–4 h) is maintained particularly by neutrophil infiltration with production of the prostaglandins, nitric oxide and cytokines (TNF-α and IL-1β) (BoughtonSmith et al., 1993; Di Rosa and Sorrentino, 1968; Ianaro et al., 1994). However, BBL had no efficacy as anti-inflammatory against the osmotic edema elicited by dextran (Lo et al., 1982), highlighting the important role of leguminous lectins in cellular events of acute inflammatory processes (Assreuy et al., 1999; Assreuy et al., 1997; Napimoga et al., 2007; Pinto et al., 2013). Importantly, all of these effects were reversed by the association of lectins with their binding sugar, as observed for BBL, implying that the lectin domain is important for anti-inflammatory activity. The anti-inflammatory effect of BBL in carrageenan-induced paw edema was further confirmed in the peritonitis model. BBL potently attenuated the leukocyte migration induced by
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carragenan, a substance that stimulates resident macrophages to release primary cytokines (TNF-α and IL-1β) that are chemotactic to neutrophils (Souza et al., 1988). In agreement, the differential leukocyte count showed that neutrophils are the principal cells involved in the anti-inflammatory effects of BBL. In addition, intravital microscopy showed a reduction in the amount of leukocyte rolling and adhesion to the endothelium of mesenteric vessels by BBL. Similar results have been observed for the lectins isolated from Lonchocarpus sericeus (Napimoga et al., 2007) and L. auriculata (Alencar et al., 2010), which exhibit binding specificity for N-acetylglucosamine and galactose, respectively. These results were corroborated by the observation that oligosaccharide sequences with 6-O-sulfation at galactose (6′-sulfo) or at N-acetylglucosamine (6-sulfo) present in sialylLewis(x) are expressed in high-endothelial venules, which are considered the endogenous ligand for adhesion molecules, such as the endogenous lectin L-selectin (Galustian et al., 1997; Fukuda et al., 1999). In our study, galactose was capable of completely blocking the anti-inflammatory effect of BBL during leukocyte migration, suggesting that BBL could establish competitive interaction with selectins for binding sites both in endothelial and leukocyte cell membranes, preventing the rolling and, consequently, the adhesion of neutrophils. Excessive generation of MPO-derived oxidants has been linked to tissue damage in acute and chronic inflammation (van der Veen et al., 2009). Thus, the interference of BBL with neutrophil migration was also confirmed by reduced peritoneal MPO activity, a heme-containing enzyme that is abundantly expressed in neutrophils. Accordingly, this effect had been demonstrated for L. sericeus, Canavalia grandiflora and L. auriculata lectins (Napimoga et al., 2007; Nunes et al., 2009; Alencar et al., 2010). Bauhinia bauhinioides was able to reduce the peritoneal concentration of the primary cytokines TNF-α and IL-1β. TNF-α and IL-1β induce the expression of selectins, up-regulate intercellular adhesion molecules in leukocytes, endothelial and resident cells and stimulate the production and release of platelet activating factor, leukotriene B4 and chemokines, mediators that induce neutrophil recruitment (Wagner; Roth, 2000). In addition to the decrease in cytokine levels in the peritoneal fluid, BBL inhibited the cell migration induced by TNF-α, suggesting that lectin interferes not only in the synthesis but also in the interaction of TNF-α with its receptor. Moreover, the signaling events triggered by TNF-α in the inflammatory process could be a possible target of BBL because a lectin-like domain of TNF-α has been shown to be implicated in its biological activities on mammalian cells via TNFR1 and TNFR2 (Lucas et al., 1997; Lucas et al., 1994). BBL may also act on Toll-like receptors because the activation of innate immunity by λ-carrageenan depends Toll-like receptor-4 via IL-6 and TNF-α induction (Tsuji et al., 2003). Further, galactose-binding lectins exhibit higher affinity for interaction with Toll-like receptor-4 (Unitt and Hornigold, 2011). Therefore, there are many other possible targets for the BBL anti-inflammatory effect. Nevertheless, the present study indicates the potential therapeutic use of leguminous lectins and provides tools for better understanding the glycobiology aspects of inflammation. In conclusion, the present investigation demonstrates that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration through the reduction of TNF-α and IL-1 via the lectin domain.
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2015; 28: 285–292
THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides
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J. Mol. Recognit. 2015; 28: 285–292
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Alencar NM, Teixeira EH, Assreuy AM, Cavada BS, Flores CA, Ribeiro RA. 1999. Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment. Mediators Inflamm. 8(2): 107–113. doi: 10.1080/09629359990603 Alencar NM, Cavalcante CF, Vasconcelos MP, Leite KB, Aragao KS, Assreuy AM, Nogueira NA, Cavada BS, Vale MR. 2005. Anti-inflammatory and antimicrobial effect of lectin from Lonchocarpus sericeus seeds in an experimental rat model of infectious peritonitis. J. Pharm. Pharmacol. 57(7): 919–922. doi: 10.1211/0022357056352 Alencar NM, Oliveira RS, Figueiredo JG, Cavalcante IJ, Matos MP, Cunha FQ, Nunes JV, Bomfim LR, Ramos MV. 2010. An anti-inflammatory lectin from Luetzelburgia auriculata seeds inhibits adhesion and rolling of leukocytes and modulates histamine and PGE2 action in acute inflammation models. Inflamm. Res. 59(4): 245–254. doi: 10.1007/s00011-009-0092-9 Altschul SF, Madden TL, Schaffer 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): 3389–3402. Assreuy AM, Shibuya MD, Martins GJ, De Souza ML, Cavada BS, Moreira RA, Oliveira JT, Ribeiro RA, Flores CA. 1997. Anti-inflammatory effect of glucose-mannose binding lectins isolated from Brazilian beans. Mediators Inflamm. 6(3): 201–210. doi: 10.1080/09629359791695 Assreuy AM, Martins GJ, Moreira ME, Brito GA, Cavada BS, Ribeiro RA, Flores CA. 1999. Prevention of cyclophosphamide-induced hemorrhagic cystitis by glucose-mannose binding plant lectins. J. Urol. 161(6): 1988–1993. Bilgin M, Burgazli KM, Rafiq A, Mericliler M, Neuhof C, Oliva ML, Parahuleva M, Soydan N, Doerr O, Abdallah Y, Erdogan A. 2014. Effect of Bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration. Eur. Rev. Med. Pharmacol. Sci. 18(1): 46–51. Boughton-Smith NK, Deakin AM, Follenfant RL, Whittle BJ, Garland LG. 1993. Role of oxygen radicals and arachidonic acid metabolites in the reverse passive Arthus reaction and carrageenin paw oedema in the rat. Br. J. Pharmacol. 110(2): 896–902. Bradley PP, Priebat DA, Christensen RD, Rothstein G. 1982. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Invest. Dermatol. 78(3): 206–209. Brito MV, de Oliveira C, Salu BR, Andrade SA, Malloy PM, Sato AC, Vicente CP, Sampaio MU, Maffei FH, Oliva ML. 2014. The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models. Thromb. Res. 133(5): 945–951. doi: 10.1016/j.thromres.2014.02.027 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(2): 123–135. Cechinel Filho V. 2009. Chemical composition and biological potential of plants from the genus Bauhinia. Phytother. Res. 23(10): 1347–1354. doi: 10.1002/ptr.2756 Di Rosa M, Sorrentino L. 1968. The mechanism of the inflammatory effect of carrageenin. Eur. J. Pharmacol. 4(3): 340–342. Di Rosa M, Giroud JP, Willoughby DA. 1971. Studies on the mediators of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine. J. Pathol. 104(1): 15–29. doi: 10.1002/ path.1711040103 Ferreira SH, Moncada S, Parsons M, Vane JR. 1974. Proceedings: the concomitant release of bradykinin and prostaglandin in the inflammatory response to carrageenin. Br. J. Pharmacol. 52(1): 108p–109p. Fortes ZB, Farsky SP, Oliveira MA, Garcia-Leme J. 1991. Direct vital microscopic study of defective leukocyte–endothelial interaction in diabetes mellitus. Diabetes 40(10): 1267–1273. Fukuda M, Hiraoka N, Yeh JC. 1999. C-type lectins and sialyl Lewis X oligosaccharides. Versatile roles in cell–cell interaction. J. Cell Biol. 147(3): 467–470. Galustian C, Lawson AM, Komba S, Ishida H, Kiso M, Feizi T. 1997. SialylLewis(x) sequence 6-O-sulfated at N-acetylglucosamine rather than at galactose is the preferred ligand for L-selectin and de-N-acetylation of the sialic acid enhances the binding strength. Biochem. Biophys. Res. Commun. 240(3): 748–751.
D. K. F. B. GIRÃO ET AL. characterization of a new pro-inflammatory lectin from Bauhinia bauhinioides Mart (Caesalpinoideae) seeds. Protein Pept. Lett. 18(4): 396–402. Silva HC, Bari AU, Rocha BA, Nascimento KS, Ponte EL, Pires AF, Delatorre P, Teixeira EH, Debray H, Assreuy AM, Nagano CS, Cavada BS. 2013. Purification and primary structure of a mannose/glucose-binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti-inflammatory properties. J. Mol. Recognit. 26(10): 470–478. doi: 10.1002/jmr.2289 Simoes RC, Rocha BA, Bezerra MJ, Barroso-Neto IL, Pereira-Junior FN, da Mata Moura R, do Nascimento KS, Nagano CS, Delatorre P, de Freitas Pires A, Assreuy AM, Sampaio AH, Cavada BS. 2012. Protein crystal content analysis by mass spectrometry and preliminary X-ray diffraction of a lectin from Canavalia grandiflora seeds with modulatory role in inflammation. Rapid Commun. Mass Spectrom. 26(7): 811– 818. doi: 10.1002/rcm.6171 de Souza GE, Ferreira SH. 1985. Blockade by antimacrophage serum of the migration of PMN neutrophils into the inflamed peritoneal cavity. Agents Actions 17(1): 97–103. Souza GE, Cunha FQ, Mello R, Ferreira SH. 1988. Neutrophil migration induced by inflammatory stimuli is reduced by macrophage depletion. Agents Actions 24(3-4): 377–380.
Souza MA, Carvalho FC, Ruas LP, Ricci-Azevedo R, Roque-Barreira MC. 2013. The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties. Glycoconj. J. 30(7): 641–657. doi: 10.1007/s10719-012-9464-4 Tsuji RF, Hoshino K, Noro Y, Tsuji NM, Kurokawa T, Masuda T, Akira S, Nowak B. 2003. Suppression of allergic reaction by lambdacarrageenan: toll-like receptor 4/MyD88-dependent and independent modulation of immunity. Clin. Exp. Allergy 33(2): 249–258. Unitt J, Hornigold D. 2011. Plant lectins are novel Toll-like receptor agonists. Biochem. Pharmacol. 81(11): 1324–1328. doi: 10.1016/j. bcp.2011.03.010 van der Veen BS, de Winther MP, Heeringa P. 2009. Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease. Antioxid. Redox Signal. 11(11): 2899–2937. doi: 10.1089/ars.2009.2538 Wagner JG, Roth RA. 2000. Neutrophil migration mechanisms, with na emphasis on the pulmonar vasculature. Pharmacological Reviews Bethesda 52(3): 349–374.
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J. Mol. Recognit. 2015; 28: 285–292
Revised: 3 October 2014,
Accepted: 4 October 2014,
Published online in Wiley Online Library: 23 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2441
The galactose-binding lectin isolated from Bauhinia bauhinioides Mart seeds inhibits neutrophil rolling and adhesion via primary cytokines Deysen Kerlla Fernandes Bezerra Girãoa, Benildo Sousa Cavadab, Alana de Freitas Piresc, Timna Varela Martinsc, Álvaro Xavier Francoa, Cecília Mendes Moraisa, Kyria Santiago do Nascimentob, Plinio Delatorred, Helton Colares da Silvab, Celso Shiniti Naganoe, Ana Maria Sampaio Assreuyc and Pedro Marcos Gomes Soaresa* 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. Keywords: Bauhinia bauhinioides; galactose-binding lectin; anti-inflammatory effect; neutrophil; TNF-α
INTRODUCTION
to their effects on inflammatory processes. The anti-inflammatory effect of plant lectins has been shown to be primarily because of
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* Correspondence to: P. Marcos Gomes Soares, Departamento de Morfologia, Universidade Federal do Ceará, Rua Delmiro de Farias s/n, 60430-170, Fortaleza, Ceará, Brazil. E-mail: [email protected] a D. K. F. B. Girão, Á. X. Franco, C. M. Morais, P. M. G. Soares Departamento de Morfologia, Universidade Federal do Ceará, Rua Delmiro de Farias s/n, 60430-170, Fortaleza, Ceará, Brazil b B. S. Cavada, K. Santiago do Nascimento, H. C. Silva Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 907, Box 6043, 60440-970, Fortaleza, Ceará, Brazil c A. Pires, T. V. Martins, A. M. S. Assreuy Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, Av. Paranjana 1700, 60740-000, Fortaleza, Ceará, Brazil d P. Delatorre Departamento de Biologia Molecular, Universidade Federal da Paraíba, Cidade Universitária, 58059-900, João Pessoa, Brazil e C. S. Nagano Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Av. Mister Hull s/n, Bloco 827, Fortaleza, Brazil
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Legumes in the genus Bauhinia (Caesalpinoideae) are widely distributed in most of the tropical regions worldwide including Africa, Asia and South America (Cechinel Filho, 2009). Many biological activities have been identified in compounds from Bauhinia bauhinioides (BBL), being one of the most studied antithrombotic and antitumor effects resulting from the action of kallikrein isolated from this bean (Brito et al., 2014; Bilgin et al., 2014). Another important substance extracted from BBL is a sugar-binding protein, denominated lectin. The property of specifically and reversibly binding to carbohydrates or precipitate glycoconjugates is a feature that enables lectins to interact with a variety of mammalian cells, modulating several biological events, including the inflammatory process (Sharon and Lis, 2004; Souza et al., 2013). In fact, the lectin domain of selectins plays a pivotal role in leukocyte arresting and rolling through reversible binding to complex carbohydrates (Kansas, 1996; Malik and Lo, 1996). However, the mechanism by which plant lectins interfere with the inflammatory process has not yet been elucidated. Despite differences in carbohydrate-binding specificity and quaternary structures, legume lectins present high sequential identity (Cavada et al., 2001) but differ in potency and efficacy with respect
D. K. F. B. GIRÃO ET AL. Table 1. Amino acid sequences and molecular masses of Bauhinia bauhinioides peptides obtained by tandem mass spectrometry Peptidea T1 T2 T2′ T3 T4 T5 T6 T7 T8
Experimental mass (Da)
Theoretical mass (Da)
Sequence
1313.7383 1436.7736 1464.7684 1870.8809 803.4145 1902.9476 1596.7823 1966.8909 2525.0498
1313.6444 1436.6044 1464.7644 1870.9201 803.4089 1902.7843 1596.7644 1966.8043 2525.1343
AXFXAPPDFPVK HXGXNVNSXESVR HXGXNVNDXESVR AXYVAPXHXWDDTDSR NSXESVR YXXSWSFSSTTTNXQR TDSFYTTFTFVXR DVFSESVATAHVGYDNEK WEDKDVFSESVATAHVGYDNEK
a Peptide obtained from cleavage with trypsin (T). In peptide sequence, X represents leucine or isoleucine residues, which cannot be distinguished by mass.
interference with neutrophil migration via the inhibition of primary cytokines (TNF-α and IL1-β) and interaction with the lectin domain (Assreuy et al., 1997; Assreuy et al., 1999; Mota et al., 2006; Napimoga et al., 2007; Pinto et al., 2013). Most of these studies were performed in rodents via systemic treatment with lectins that displays binding specificity for glucose–mannose (Assreuy et al., 1999; Assreuy et al., 1997; Rocha et al., 2011; Simoes et al., 2012; Pinto et al., 2013; Silva et al., 2013) and N-acetylglucosamine (Alencar et al., 1999; Alencar et al., 2005; Napimoga et al., 2007). However, the anti-inflammatory effect of galactose-binding lectins has only been demonstrated for those isolated from the leguminous Luetzelburgia auriculata (Alencar et al., 2010). The lectin of BBL, which displays binding affinity for galactose, has a molecular mass of 28.310 Da as demonstrated by mass spec-
trometry. BBL lectin exhibits hemagglutinating activity against rabbit erythrocytes and edematogenic effects in rats through carbohydrate site interaction (Silva et al., 2011). Thus, the present study aimed to reveal the partial amino acid sequence of the lectin isolated from the leguminous BBL and to investigate whether this lectin possesses anti-inflammatory properties in acute models of inflammation in rats.
MATERIALS AND METHODS Drugs and reagents Dextran, carrageenan, TNF-α, ammonium bicarbonate, D-galactose, formic acid, o-dianosidine dihydrochloride and [Glu1]-fibrinopeptide B were purchased from Sigma (St. Louis, MO, USA); acetonitrile was
A
B
286
Figure 1. Mass spectra of Bauhinia bauhinioides (BBL) isoform peptide sequences. Collision-induced dissociation of two isoforms of T2 ion. (A) A doubly charged ion at m/z 719.83 with sequence-specific y-ion series interpreted as HXGXNVNSXESVR and (B) a doubly charged ion at m/z 733.39 with sequence-specific y-ion series interpreted as HXGXNVDSXESVR. The letter X in peptide sequence represents leucine or isoleucine residues, which cannot be distinguished by mass.
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J. Mol. Recognit. 2015; 28: 285–292
THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides purchased from Tedia (Rio de Janeiro, Brazil); trypsin from Promega (Madison, WI, USA); sterile saline 0.9% was purchased from Fresenius Kabi Brasil (Aquiraz, Ceará, Brazil); 5-5′-dithiolbis2-nitrobenzoic acid and Tris-buffer were purchased from Dinâmica Química Contemporânea Ltda (Diadema, São Paulo, Brazil); 2,2,2-tribromoethanol (Avertin®) was purchased from Chemada Fine Chemicals Company Ltda (Nir Itzhak, D.N. HaNegvev, Israel); and kits for ELISA (TNF-α and IL-1β) were obtained from R&D Systems (Minneapolis, MN, USA). All other drugs and reagents were of analytical grade. Partial amino acid sequence Proteolytic cleavage Bauhinia bauhinioides was submitted to SDS-PAGE (Laemmli, 1970), and the bands were excised, bleached with 50 mM ammonium bicarbonate in 50% acetonitrile, dehydrated in 100% acetonitrile and dried in a Speedvac (LabConco, Kansas City, MO, USA). For proteolytic cleavage, gels were rehydrated in 50 mM ammonium bicarbonate containing trypsin at 37 °C overnight. The obtained peptides were extracted in 50% acetonitrile containing 5% formic acid and concentrated in Speedvac.
Tryptic peptides were separated in BEH300 C18 columns (100 μm × 100 mm) using the nanoAcquity™ (Waters Technologies of Brazil Ltda, Barueri, São Paulo, Brazil) system and eluted at 600 μL/min with acetonitrile gradient (10–85%) containing 0.1% formic acid. The liquid chromatography system was connected to a nanoelectrospray mass spectrometer source (SYNAPT HDMS system, Waters Corp., Milford, MA, USA). The mass spectrometer was operated in positive mode using a source temperature of 90 °C and capillary voltage of 3.5 kV. The instrument was calibrated with fragments of the double protonated ion [Glu1]-fibrinopeptide B (m/z 785.84), and the Lock mass used during the acquisition was the intact ion. The LC–MS/MS procedure was performed according to the data dependent acquisition function, selecting MS/MS doubly or triply charged precursor ions. Ions were fragmented by collision-induced dissociation using argon as the collision gas and ramp collision energy that varied according to the charge state of the selected precursor ion. Data acquisition was performed at an m/z range of 400–2100 for the MS survey and at an m/z range of 50–3000 for MS/MS. Data were processed with MassLynx 4.0 software (Waters Corp.). The collision-induced dissociation spectra were interpreted manually using the Peptide Sequencing tool in MassLynx 4.0 software. BBL peptide sequences were compared
B
A 1,5
*
0,9
1,2
*
*
#
#
* 0,6
Edema (ml)
Saline Cg (1%) Cg + BBL (0.01mg/kg) Cg + BBL (0.1mg/kg) Cg + BBL (1mg/kg)
1,2
Edema (ml)
Tandem mass spectrometry
Saline Cg (1%) Cg + BBL (1mg/kg) Cg + BBL (1mg/kg) + galactose [0.1M] Cg + galactose [0.1M] Cg + Indo (10mg/kg)
0,9
* 0,6
#
#
a
a
* 0,3
0,3
*
a
0,0
0,0 0
1
2
3
4
0
1
2
Time(h)
3
4
Time (h)
C Saline Cg (1%) Cg +BBL + galactose
Cg + BBL (1 mg/kg) galactose (0.1 M)
AUC (arbitrary units)
4 First phase
Second phase
**
3
*
2
# 1
*
0
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Figure 2. The inhibitory effect of Bauhinia bauhinioides (BBL) on the paw edema induced by carrageenan. Animals were treated intravenously with saline, D-galactose, BBL or BBL (1 mg/kg) with D-galactose (0.1 M) or subcutaneous indomethacin (indo) (10 mg/kg), 30 min before carrageenan (Cg). Edema was measured at 30 min, 1, and 2–4 h after Cg and is expressed as the variation in paw volume (ml) or area under curve (AUC) (arbitrary units) in relation to baseline. (A) Dose response curve, (B) reversal of the effect of BBL by incubation with ligand sugar and (C) AUC first and second phase. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg, **p < 0.05 versus Cg + BBL, “a” p < 0.05 Cg versus Cg + indo.
D. K. F. B. GIRÃO ET AL. with all of the nonredundant proteins deposited in the National Center of Biotechnology Information using BLAST (Altschul et al., 1997), and the proteins with the best e-values were selected for sequence alignments at ClustalW.
administration of the inflammatory agents. Positive control was indomethacin (10 mg/kg, s.c.).
Experimental models of inflammation
Peritonitis was induced by intraperitoneal (i.p.) injection of carrageenan (1 mg) or TNF-α (0.1 ηg) 30 min after the intravenous treatment with BBL (1 mg/kg). Negative controls received sterile saline (i.p.). After 4 h, peritoneal fluid was collected in 10 ml of saline (5 IU heparin) for total and differential leukocyte counts, which are expressed per ml (de Souza and Ferreira, 1985). The peritoneal fluid stimulated with carrageenan was centrifuged (3000 g × 15 min), and the obtained supernatant was assessed for the content of the cytokines TNF-α and IL-1β (pg/dl) by enzyme-linked immunosorbent assay and myeloperoxidase (MPO) activity (U/ml) (Bradley et al., 1982). The effect of BBL (1 mg/kg) was also evaluated for leukocyte rolling and adhesion by intravital microscopy.
Animals Wistar rats (150–200 g) were kept in cages (six in each) in a controlled environment (circadian cycle, 25 °C, with food and water ad libitum). Experimental protocols were previously approved by the Institutional Animal Care and Use Committee of the State University of Ceará (No. 10130208/40) following the recommendations of the Guide for the Care and Use of Laboratory Animals of the US Department of Health and Human Services (NIH publication No. 85-23, revised 1985). Paw edema Paw volume was measured immediately before (time zero) subcutaneous (s.c.) injection of inflammatory stimuli (carrageenan or dextran; 300 μg/paw) into the hind paw of rats and thereafter at selected time intervals (0.5, 1–4 h) by hydroplethysmometry (Panlab LE 7500, Barcelona, Spain). The results are expressed as the variation in paw volume (ml) in relation to baseline. The area under the time-course curve (AUC) was expressed in arbitrary units (Landucci et al., 1995). BBL (0.01, 0.1 e 1 mg/kg) or saline was administered by intravenous route 30 min prior to the
A
Peritonitis
Intravital microscopy Leukocyte adhesion and rolling were evaluated 4 h after the induction of peritonitis with carrageenan. The animals were anesthetized with tribromoethanol (250 mg/kg, i.p.) before exposition of the mesenteric bed for in situ microscopic examination. Leukocytes rolling was defined as white blood cells that move at a velocity significantly slower than that of erythrocytes in a given microvessel. Leukocytes were considered to
B
Total
Mononuclear
Neutrophil
Eosinophil Total
Neutrophil Eosinophil
*
6,000
**
*
**
#
*
10,000
4,000
2,000
12,000
Mononuclear
Leukocytes/mL
Leukocytes/mL
8,000
# 8,000
*
6,000 4,000
#
2,000
#
0
0 galactose [0.1M] BBL (1mg/Kg)
Saline
Saline
TNF-
(0.1 ng) BBL
Cg (1mg/cavity)
D
C 35
Saline
Cg + BBL
Cg
Cg + BBL + galactose
30
*
**
25 20 15 10 5
2.0
#
Leukocyte adhesion/min
Leukocyte rolling/min
40
1.5
Saline Cg
Cg + BBL Cg + BBL + galactose
* **
1.0
0.5
#
288
Figure 3. The inhibitory effect of Bauhinia bauhinioides (BBL) on leukocyte migration. Animals were treated intravenously with saline, D-galactose (0.1 M), BBL (1 mg/kg) or BBL with D-galactose 30 min before inflammatory stimuli. Total and differential cell counts were performed 4 h after the induction of peritonitis with (A) carrageenan (Cg; 1 mg) or (B) TNF-α (0.1 ηg). The number of (C) rolled and (D) adherent leukocytes were evaluated in mesenteric microcirculation 4 h after Cg by intravital microscopy. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg, **p < 0.05 versus Cg + BBL.
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THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides be adherent to the venular endothelium if they remained stationary for more than 30 s. Rolling and adherent cells were counted for 10 min in a 10 μm segment of postcapillary venules (three per animal, diameter 10–18 μm) and expressed as the number of rolled leukocytes/min and adherent cells/100 μm2 (Fortes et al., 1991).
significantly decreased leukocyte rolling by 58% (11.78 ± 0.547) and adhesion by 68% (0.385 ± 0.066 cells/min) (Figure 3(C, D)). The association of BBL with its carbohydrate ligand galactose completely abolished the inhibitory effect of BBL in the leukocyte migration induced by Cg (Figure 3(A,C,D)).
Lectin and carbohydrate interaction assay
Bauhinia bauhinioides inhibits the increase of inflammatory mediators
Bauhinia bauhinioides (1 mg/kg) was incubated with its binding sugar (D-galactose; 0.1 M) for 1 h at 37 °C to allow lectin–sugar interaction before performing the inflammatory protocols. Lectin and sugar were also incubated in separated solutions under the same conditions as controls.
Carrageenan significantly increased MPO activity (0.73 ± 1.72 U/ml), TNF-α (267.0 ± 29.04 ρg/ml) and IL-1β (632.2 ± 34.25 ρg/ml) levels, as determined in the peritoneal exudate 4 h after challenge. BBL significantly inhibited MPO by 84% (0.12 ± 0.92 U/ml), TNF-α by 68% (85.5 ± 16.3 ρg/ml) and IL-1β by 47% (330.6 ± 39.98 ρg/ml) (Figure 4).
Statistical analysis Statistical analyses and the construction of graphs were performed using the software GraphPad Prism® (GraphPad Software, San Diego, CA, USA). Comparisons were determined by analysis of variance, followed by Bonferroni’s test. Values at p < 0.05 were considered significant. Data are expressed as the mean ± SEM (n = 6).
RESULTS Amino acid sequence analysis The partial amino acid sequence of BBL, which was obtained by MS/MS after proteolysis of sodium dodecyl sulfatepolyacrylamide gel electrophoresis bands, revealed eight peptides, resulting in a total of 86 amino acid residues (Table 1). Sequence heterogeneity was observed in one peptide D/S (aspartic acid/serine) (T2/T2′), indicating that BBL preparations contain highly homologous isolectins (Figure 1). Bauhinia bauhinioides anti-inflammatory effect
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Figure 4. Bauhinia bauhinioides (BBL) reduced myeloperoxidade (MPO) activity and cytokine (TNF-α and IL-1β) levels in peritoneal fluid. Animals were injected intravenously with saline or BBL 30 min prior to intraperitoneal injection of carrageenan (Cg). (A) MPO, (B) TNF-α and (C) IL-1β levels were determined by enzyme-linked immunosorbent assay. Mean ± SEM (n = 6). Analysis of variance and Bonferroni’s test; *p < 0.05 versus saline, #p < 0.05 versus Cg.
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Carrageenan induced a progressive and intense paw edema that was initiated during the first hour, reached maximal values between 3 and 4 h and was reduced by BBL at 1 mg/kg by 21% (3 h) and 19% (4 h) (Figure 2(A)). The anti-inflammatory effect of BBL (AUC: 1.30 ± 0.008), observed only in the second phase of carrageenan-induced edema (AUC: 2.02 ± 0.006), was completely blocked by BBL (Figure 2(B)) in association with galactose (AUC: 2.82 ± 0.21). Galactose per se did not induce any effect (Figure 2(C)). Indomethacin, the positive control, inhibited the carrageenan-induced edema in all time after second hour (Figure 2(B)). In contrast, the osmotic edema elicited by dextran (AUC: 1.191 ± 0.115) was not altered by BBL (AUC: 1.10 ± 0.749) (figure not shown). Moreover, BBL (1 mg/kg) also exhibited inhibitory effects in the peritonitis model. BBL inhibited the leukocyte migration induced by carrageenan (6275 ± 712.2) by 72% (1725 ± 121.6) and by TNF-α (10375 ± 11.58) by 28% (7600 ± 942.2) (Figure 3(A,B)). This effect was more pronounced in neutrophils because BBL inhibited 51% (849.5 ± 165.7) of the neutrophil migration induced by carrageenan (1725 ± 121.6) and 64% (1683 ± 277.8) of that induced by TNF-α (4649 ± 907.2 cells/ml) (Figure 3). Intravital microscopy revealed that carrageenan induced significant increases in endothelial leukocyte rolling (28.32 ± 1.619) and adhesion (1.216 ± 0.189) compared with saline. BBL
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Interest in studying the effects of plant lectins has been increasing because glycoconjugate interactions have been implicated in inflammation pathways. Therefore, these proteins are structurally correlated and differ in the ability of binding to carbohydrates; these proteins are considered potential tools for application in biotechnology or immunotherapies (Rudiger and Gabius, 2001). In the present study, we demonstrated that BBL exhibits homology with other Bauhinia lectins and shares binding specificity for D-galactose and its derivatives (Irimura and Osawa, 1972; Silva et al., 2007; Silva et al., 2011). In fact, the partial amino acid sequence reported here corresponds to approximately 35% of the total BBL sequence. Comparisons with other sequences from the National Center of Biotechnology Information databank revealed that BBL peptides show identity from 35.87% to 39.13% with other Bauhinia lectins, such as Bauhinia purpurea (Swiss-Prot accession code: P16030.2), Bauhinia variegata (GenBank accession code: ABQ45362.1) and Bauhinia ungulata (GenBank accession code: ABD19775.1), respectively. Also, the partial amino acid sequence exhibited sequence identity to other galactose/N-acetyl galactosamine-specific lectins from the Leguminosaeae family with 44.57% for lectin III of Griffonia simplicifolia (SwissProt accession code: P24146.3), 40.22% for Vatairea macrocarpa (SwissProt accession code: P81371), 35.87% for Robinia pseudoacacia (PDB accession code: 1FNZ) and 34.78% for Sophora japonica (SwissProt accession code: P93538). Sequence heterogeneity was observed in one peptide D/S (T2/T2’), indicating that BBL preparations contain highly homologous isolectins. Similarly, the cloning and sequence of the B. variegata lectin gene have suggested that this protein could be encoded by a family of genes, which led to the identification of two isoforms (Pinto et al., 2008). The present study also demonstrated the anti-inflammatory effect of BBL in cellular events of acute experimental inflammation models, which is relevant because the anti-inflammatory effect of galactose-binding lectins has previously only been demonstrated for those isolated from the leguminous L. auriculata (Alencar et al., 2010). Bauhinia bauhinioides inhibited the paw edema induced by carrageenan, an effect that was restricted to the second phase. This edema is a biphasic temporal phenomenon characterized by an intense leukocyte influx that involves a complex network of inflammatory mediators (Di Rosa et al., 1971). The initial phase (0–2 h) is predominantly vascular, triggered by the release of biogenic amines (histamine, bradykinin and serotonin) (Ferreira et al., 1974), whereas the second phase (2–4 h) is maintained particularly by neutrophil infiltration with production of the prostaglandins, nitric oxide and cytokines (TNF-α and IL-1β) (BoughtonSmith et al., 1993; Di Rosa and Sorrentino, 1968; Ianaro et al., 1994). However, BBL had no efficacy as anti-inflammatory against the osmotic edema elicited by dextran (Lo et al., 1982), highlighting the important role of leguminous lectins in cellular events of acute inflammatory processes (Assreuy et al., 1999; Assreuy et al., 1997; Napimoga et al., 2007; Pinto et al., 2013). Importantly, all of these effects were reversed by the association of lectins with their binding sugar, as observed for BBL, implying that the lectin domain is important for anti-inflammatory activity. The anti-inflammatory effect of BBL in carrageenan-induced paw edema was further confirmed in the peritonitis model. BBL potently attenuated the leukocyte migration induced by
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carragenan, a substance that stimulates resident macrophages to release primary cytokines (TNF-α and IL-1β) that are chemotactic to neutrophils (Souza et al., 1988). In agreement, the differential leukocyte count showed that neutrophils are the principal cells involved in the anti-inflammatory effects of BBL. In addition, intravital microscopy showed a reduction in the amount of leukocyte rolling and adhesion to the endothelium of mesenteric vessels by BBL. Similar results have been observed for the lectins isolated from Lonchocarpus sericeus (Napimoga et al., 2007) and L. auriculata (Alencar et al., 2010), which exhibit binding specificity for N-acetylglucosamine and galactose, respectively. These results were corroborated by the observation that oligosaccharide sequences with 6-O-sulfation at galactose (6′-sulfo) or at N-acetylglucosamine (6-sulfo) present in sialylLewis(x) are expressed in high-endothelial venules, which are considered the endogenous ligand for adhesion molecules, such as the endogenous lectin L-selectin (Galustian et al., 1997; Fukuda et al., 1999). In our study, galactose was capable of completely blocking the anti-inflammatory effect of BBL during leukocyte migration, suggesting that BBL could establish competitive interaction with selectins for binding sites both in endothelial and leukocyte cell membranes, preventing the rolling and, consequently, the adhesion of neutrophils. Excessive generation of MPO-derived oxidants has been linked to tissue damage in acute and chronic inflammation (van der Veen et al., 2009). Thus, the interference of BBL with neutrophil migration was also confirmed by reduced peritoneal MPO activity, a heme-containing enzyme that is abundantly expressed in neutrophils. Accordingly, this effect had been demonstrated for L. sericeus, Canavalia grandiflora and L. auriculata lectins (Napimoga et al., 2007; Nunes et al., 2009; Alencar et al., 2010). Bauhinia bauhinioides was able to reduce the peritoneal concentration of the primary cytokines TNF-α and IL-1β. TNF-α and IL-1β induce the expression of selectins, up-regulate intercellular adhesion molecules in leukocytes, endothelial and resident cells and stimulate the production and release of platelet activating factor, leukotriene B4 and chemokines, mediators that induce neutrophil recruitment (Wagner; Roth, 2000). In addition to the decrease in cytokine levels in the peritoneal fluid, BBL inhibited the cell migration induced by TNF-α, suggesting that lectin interferes not only in the synthesis but also in the interaction of TNF-α with its receptor. Moreover, the signaling events triggered by TNF-α in the inflammatory process could be a possible target of BBL because a lectin-like domain of TNF-α has been shown to be implicated in its biological activities on mammalian cells via TNFR1 and TNFR2 (Lucas et al., 1997; Lucas et al., 1994). BBL may also act on Toll-like receptors because the activation of innate immunity by λ-carrageenan depends Toll-like receptor-4 via IL-6 and TNF-α induction (Tsuji et al., 2003). Further, galactose-binding lectins exhibit higher affinity for interaction with Toll-like receptor-4 (Unitt and Hornigold, 2011). Therefore, there are many other possible targets for the BBL anti-inflammatory effect. Nevertheless, the present study indicates the potential therapeutic use of leguminous lectins and provides tools for better understanding the glycobiology aspects of inflammation. In conclusion, the present investigation demonstrates that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration through the reduction of TNF-α and IL-1 via the lectin domain.
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J. Mol. Recognit. 2015; 28: 285–292
THE GALACTOSE-BINDING LECTIN ISOLATED FROM Bauhinia bauhinioides
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