Mannose-Specific Legume Lectin from the Seeds of Dolichos Lablab

Process Biochemistry 49 (2014) 529–534

Contents lists available at ScienceDirect

Process Biochemistry

jo urnal homepage: www.elsevier.com/locate/procbio

Mannose-speciﬁc legume lectin from the seeds of Dolichos lablab

(FRIL) stimulates inﬂammatory and hypernociceptive processes in mice

a,b c

Claudener Souza Teixeira , Ana Maria Sampaio Assreuy ,

a c

Vinícius José da Silva Osterne , Renata Morais Ferreira Amorim ,

c d e f

Luiz André Cavalcante Brizeno , Henri Debray , Celso Shiniti Nagano , Plinio Delatorre ,

a,e a

Alexandre Holanda Sampaio , Bruno Anderson Matias Rocha ,

a,∗

Benildo Sousa Cavada

Laboratório de Moléculas Biologicamente Ativas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil

Unidade Descentralizada de Campos Sales, Universidade Regional do Cariri, Crato, CE, Brazil

Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, Fortaleza, Ceará, Brazil

d ◦

Laboratoire de Chimie Biologique et Unité Mixte de Recherche N 8576 du CNRS, Université des Sciences et Technologies de Lille, Lille, France

Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Fortaleza, Ceará 60440-970, Brazil

Departamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza – Campus I, Universidade Federal da Paraíba, João Pessoa, PB, Brazil

r t i c l e i n f o a b s t r a c t

Article history: Lectins are proteins that speciﬁcally bind to carbohydrates and form complexes with molecules and bio-

Received 21 October 2013

logical structures containing saccharides. The FRIL (Flt3 receptor interacting lectin) is a dimeric two-chain

Received in revised form ␣␤

( )2 lectin presenting 67 kDa molecular mass. The interaction of FRIL with carbohydrates, as determined

28 December 2013

by molecular docking, showed that this lectin has highest afﬁnity to the carbohydrate trimannoside

Accepted 28 December 2013 −

( 135.618 MDS), followed by trehalosamine (−127.072 MDS) and ␣␣-trehalose (−121.729 MDS). FRIL

Available online 8 January 2014

evoked dose-dependent paw edema, increasing animal paw volumes. The edematogenic effect of FRIL

was paralleled by an increase in vascular permeability, about 10-fold higher compared to control. FRIL also

Keywords:

significantly raised the animals flinch reaction in the first, third and fifth hours in response to mechanical

Dolichos lablab lectin

␣ d

FRIL stimulation. Injection of -methyl- -mannoside associated with FRIL inhibited edema and hypernoci-

Trimannoside ception. The histopathological analysis of animal paws showed a characteristically acute inﬂammatory

Molecular docking process that included severe inﬁltration of mixed leukocytes, changes in cytoarchitecture, edema and

Pro-inﬂammatory focal areas of hemorrhage. In addition, in silico assays conﬁrmed that FRIL preferentially interacts with

trimannoside that makes up the core N-glycans cell. Therefore, our study supports the hypothesis that

the lectin domain and the likely glycoconjugates containing ␣-d-methyl mannoside residues contribute

to the inﬂammatory effects of FRIL.

1. Introduction identity [3]. Lectins with this high sequence identity and struc-

tural similarities play a broad spectrum of biological roles in cell

Lectins are proteins that speciﬁcally bind to carbohydrates and communication, host defense, fertilization, development, parasite

form complexes with molecules and biological structures con- infection and tumor metastasis by deciphering glycocodes present

taining saccharides, without altering the covalent structure of the in the structure of soluble and integral cell membrane glycoconju-

glycosyl ligands [1,2]. The structure of legume lectins is either gates [4,5].

dimeric or tetrameric, and their subunits reveal high structural Experimental models have demonstrated that plant lectins are

able to trigger many events related to inﬂammatory processes in

vitro, such as lymphocyte proliferation [6], histamine release from

∗ mast cells [7,8], production of nitric oxide and apoptosis [9,10].

Corresponding author at: Departamento de Bioquímica e Biologia Molecular,

In vivo, local application of some plant lectins elicits paw edema,

Universidade Federal do Ceará, BioMol-Lab, Campus do Pici S/N, 60440-970 Forta-

increased vascular permeability and neutrophil migration [11–13],

leza, Ceará, Brazil. Tel.: +55 85 33669818; fax: +55 85 33669818.

E-mail address: [email protected] (B.S. Cavada). while systemic application inhibits such events [14,15].

530 C.S. Teixeira et al. / Process Biochemistry 49 (2014) 529–534

Similar to other legume lectins, those from the Phaseoleae 2.4. Afﬁnity assays

tribe produce either anti-inﬂammatory or proinﬂammatory effects

Afﬁnity assays were performed by serial dilution employing rabbit erythro-

[16,17], depending on the administration route. It has been pro-

cytes, either native or treated with proteolytic enzymes (trypsin or papain),

posed that the anti-inﬂammatory effects elicited by legume lectins

expressed as one hemagglutinating unit (HU), and deﬁned as the minimal amount

result from competitive blocking of glycosylated selectin bind- (mg) of protein per mL able to induce visible agglutination. Lectin carbohydrate-

ing sites in the membranes of leukocytes and/or endothelial cells binding speciﬁcity was assessed by the minimal inhibitory sugar concentration

[12,13]. giving full inhibition of agglutination. Twofold serial dilutions (100 mM initial

concentration) of the sugars d-glucose, d-galactose, D-mannose, D-arabinose,

Dolichos lablab (Phaseolinae subtribe) is encountered as distinct

D-fructose, N-acetyl-d-glucosamine, ␣-methyl-D-galactopyranoside, ␣-methyl-D-

varieties. D. lablab var. typicus (Indian beans), D. lablab var. lignosus

glucopyranoside, saccharose, ␣- and ␤-lactose were prepared in 150 mM NaCl. The

(ﬁeld beans), (D. lablab var. rongai) and their seeds contain Man/Glc- lectin (4 HU) was added to each dilution.

and Gal/GalNac-speciﬁc lectins [18].

2.5. Molecular mass determination

The Man/Glc-speciﬁc lectin of Dolichos lablab (hyacinth bean)

(hereinafter termed FRIL, Flt3 receptor interacting lectin) is a

SDS–polyacrylamide gel electrophoresis was carried out on slabs of 12% poly-

␣␤

dimeric two-chain ( )2 lectin presenting 67 kDa molecular mass. acrylamide in the absence or presence of ␤-mercaptoethanol (2%) using the

FRIL has similar afﬁnity for monosaccharides, disaccharides, and Mini-PROTEAN II multiscreen apparatus (Bio-Rad; Milan, Italy), and the protein

bands were stained with Coomassie Brilliant Blue R-250. The apparent molecular

trisaccharides. The recognition of ␣-methyl-mannoside is quite

mass was assessed using standard proteins of known molecular mass.

similar to ␣␣-trehalose, trehalosamine, and the trimannoside core

of N-glycans [19]. Glycans, which are present in the cell surfaces,

2.6. Molecular docking

play crucial roles in various physiological events involving cell

surface recognition [20]. In particular, cell N-glycans containing The crystal structure of FRIL monomer (PDB code 1QM0) was used for all docking

simulations. The 3D carbohydrate structures were drawn using the online software

this trimannoside core are involved in many processes of cell–cell

PRODRG [27]. The MD was performed using the Molegro Virtual Docker (MVD)

recognition, such as leukocyte recruitment [21].

software [28]. Moldock score [GRID] was calculated using scoring function. Grid

In vitro, FRIL seems to have a protective role in neural and

resolution was 0.30 A˚ with radius of 15. The search algorithm used was Moldock

hematopoietic stem cells, inhibiting cell proliferation and differ- Optimizer with default settings. The number of runs was 10, and the maximum

interactions were 2000. The population size and maximum number of pose were

entiation [22,23] via downregulation of cyclin D3 and CDK6 and

200 and 10, respectively. The protein–ligand interaction energy was expressed in

activation of P53 [24]. However, to date, no study has reported the

the form of a Moldock score (MDS) [28] in arbitrary units. A more negative value

effect of FRIL on the in vivo inﬂammatory process and its correlation

reﬂects a stronger interaction.

with cell glycans.

Therefore, the aim of this study was to investigate the effect of 2.7. Edema and vascular permeability

FRIL on inﬂammation and hypernociception using mouse models

Paw edema was induced by subcutaneous (s.c.) intraplantar injection of FRIL (3,

and investigate lectin afﬁnity for carbohydrates by conventional

30 and 300 ␮g/paw) and measured from 0.5 to 72 h using a plethysmometer (PanLab

methods in order to associate these data with molecular docking

LE 7500, Spain). The control group received sterile saline (NaCl 0.9%; 0.01 mL/paw)

(MD). s.c. Data were expressed as the increase in paw volume (␮L) displacement through-

out the experiment subtracted from the basal volume, measured at zero time, or the

area under curve (AUC) (arbitrary units) [29].

2. Materials and methods

Vascular permeability was evaluated 5 h after application of stimulus (FRIL

300 ␮g/paw). To accomplish this, Evans Blue (25 mg/kg) was injected by intravenous

2.1. Animals

(i.v.) route 1 h before euthanasia. Paws were sectioned, weighed and incubated in

◦

␮

formamide for 72 h at 37 C. The extracted dye was estimated at A600 nm ( g Evans

Male Swiss mice (25–30 g) were maintained in a controlled environment (cir-

◦ Blue/g tissue).

cadian cycle, 25 C, food and water ad libitum). Experiments were conducted in

accordance with current guiding principles for the care and use of research ani-

2.8. Hypernociception

mals (NIH guidelines) and approved by the Ethical Committee of the Universidade

Estadual do Ceará, Brazil (UECE – no. 10130208-8/40).

Mice were placed individually in clear plexiglass boxes on elevated wire mesh

platforms to allow access to the ventral surface of the hind paws. They were fur-

ther acclimatized to the testing chambers, and the frequency of paw withdrawal

2.2. Drugs and reagents

(ﬂinch reaction) in response to 6 applications of von Frey ﬁlament (0.8 g) was deter-

mined before (basal value) and 1, 3 and 5 h after intraplantar s.c. injection of FRIL

Acrylamide, N ,N -methylenebisacrylamide, bovine serum albumin (BSA),

(300 ␮g/paw). Control group received saline (0.01 mL/paw) s.c.

Coomassie Brilliant Blue (G-250 and R-250), Evans Blue, ␭-carrageenan, ␣-methyl-

d-mannoside, d-glucose, d-galactose, d-mannose, d-arabinose, d-fructose, N-acetyl-

2.9. Lectin inhibition by ˛-methyl-d-mannoside

d-glucosamine, ␣-methyl-d-galactopyranoside, ␣-methyl-d-glucopyranoside, sac-

charose ␣- and ␤-lactose, sucrose, trypsin and papain were purchased from Sigma

FRIL (300 ␮g) was incubated with the binding sugar ␣-methyl-d-mannoside

Chemical Co., St. Louis, USA, and 2-mercaptoethanol and sodium dodecyl sulfate

◦

(0.1 M) for 60 min at 37 C before the evaluation of paw edema and hypernoci-

(SDS) were from Merck, Darmstadt, Germany. All other chemicals were of analytical

ception. Lectin and sugar were also incubated in separate solutions in the same

grade. Lectin and drugs were dissolved directly in sterile saline (0.15 M NaCl).

experimental conditions as controls.

2.3. Lectin puriﬁcation

2.10. Histology

The puriﬁcation of FRIL was performed according to Gowda et al. [25]. Mature

Histopathological analysis of the paw tissues was performed 5 h after s.c. admin-

seeds from D. lablab were ground into a ﬁne powder using a coffee mill. The pow-

istration of FRIL (300 ␮g/paw). Subplantar paw tissues were collected and ﬁxed in

der was incubated in 1:10 (w/v) 25 mM Tris–HCl buffer (THB), pH 7.5, containing

10% formaldehyde for 24 h and macroscopically analyzed. Specimens were further

◦

150 mM NaCl, at 37 C under continuous stirring for 12 h before centrifugation at

dehydrated in crescent alcoholic series, diaphanized in xylol, impregnated in paraf-

◦ ◦

10,000 × g for 20 min. The supernatant (crude extract) was precipitated with ammo-

ﬁn, melted at 60 C, blocked in parafﬁn at 37 C, sectioned (5 ␮m) by a microtome

nium sulfate (0–60%) and centrifuged, and the pellet was resuspended in 25 mM

and stained with hematoxylin-eosin (HE). The histopathological changes observed

THB, pH 7.5. The active fraction (0–60%) was submitted to afﬁnity chromatography

were as follows: edema and vessel congestion; extravasation of red blood cells;

in mannose–sepharose column, equilibrated with 25 mM THB, pH 7.5. The unbound

necrosis and inﬂammatory inﬁltrates. The morphological changes were assessed

proteins were washed with equilibrium solution, and the retained proteins were

semi-quantitatively, blinded by one independent assessor and graded as follows:

eluted with 25 mM THB, pH 7.5, containing 0.2 M D-mannose. Fractions of 1.5 mL

(0) normal tissue (no distinguishable change (0%), i.e., absence of edema, necrosis,

were collected, pooled, exhaustively dialyzed against distilled water and freeze-

inﬂammatory inﬁltrates and hemorrhagic areas); (1) discrete tissue changes (ini-

dried. Absorbance at 280 nm was used to estimate the protein concentration in all

tiation of changes, up to 30%, e.g., mild edema, necrosis, inﬂammatory inﬁltrates

chromatographic fractions. The puriﬁcation process was monitored by SDS-PAGE

and focal areas of hemorrhage); (2) moderate tissue changes (moderate changes,

[26], and the puriﬁed lectin was used for analysis and pharmacological assays.

31–60%, e.g., moderate edema, necrosis, inﬂammatory inﬁltrates and focal areas of

C.S. Teixeira et al. / Process Biochemistry 49 (2014) 529–534 531

Table 1

Puriﬁcation of FRIL (from 20 g of seed ﬂour).

a b

Steps Protein (mg/mL) Hemagglutinating activity (UH/mL) Speciﬁc activity (UH/mgP) Puriﬁcation fold (x)

Crude extract 0.35 64 182.85 1

Fraction (NH4)SO2 0–60% 0.93 512 550.5 3.01

Sepharose–mannose 0.19 512 2694.73 14.73

Protein concentration.

Haemagglutinating activity expressed in haemagglutinating units.

Table 2

hemorrhage); (3) severe tissue changes (widespread changes, 61–100%, e.g., severe

Inhibitory effect of monosaccharides and disaccharides on FRIL hemagglutinating

edema, necrosis, inﬂammatory inﬁltrates and focal areas of hemorrhage).

activity.

2.11. Statistical analysis a b c

Sugar MIC (mM) I50 (mM) MDS

d −

Histopathological data were expressed as median (maximum and minimum) -Mannose 25 1.45 62.95

d −

and analyzed by Mann–Whitney test. All other data were expressed as mean ± S.E.M. -Glucose 50 2.5–3.0 60.62

␣ d −

and analyzed by two-way ANOVA and Bonferroni test. Statistical signiﬁcance was -Methyl- -mannose 6.25 0.4 68.56

set at p < 0.05. Protocols were performed using 6–8 animals per group. N-Acetyl- -glucosamine 25 0.93 ND

d e

N-Acetyl-d-mannosamine NI NI (10) ND

Saccharose NI 9.8 ND

3. Results

␣-Lactose NI ND ND

␤

-Lactose NI ND ND

3.1. Puriﬁcation and mass determination -Galactose NI NI ND

-Xylose NI ND ND

The puriﬁcation of FRIL was achieved by ammonium sul- MIC, minimum inhibitory concentration.

Inhibition of FRIL by IgM precipitation [19].

fate precipitation, followed by afﬁnity chromatography on

Moldock score(MDS)= Einter + Eintra, where Einter is the ligand protein interaction

sepharose–mannose column (Table 1, Fig. 1A). The retained peak 2

energy: Einter = [EPLP (rij) + 332.0(qjqj/4r )].

i ∈ ligand j ∈ ligand ij

was eluted with 0.2 M D-mannose, the fractions 48–51 correspond

NI, sugar not inhibitory until a concentration of 100 mM.

to FRIL, fraction 49 being the one with highest concentration of FRIL e

No inhibition at the concentration (mM) indicated in parenthesis.

and, consequently, with higher absorption at 280 nm as shown in

Fig. 1. All the hemaggutinating activity was concentrated in this

fraction (Fig. 1A). SDS-PAGE revealed a relative mass of 67 kDa as

an inhibition assay by immunoprecipitation. Their results demon-

the protein sample was composed of four ␣ chains (14–22 kDa)

strate that the sugar ␣-methyl mannoside presented the lowest

and one ␤ chain (10 kDa), demonstrating its subunit heterogeneity

concentration able to inhibit 50% of precipitation by IgM, which

(Fig. 1B).

supports the results obtained by our group. The values of these

assays for monossacharides and dissacharides tested by Mo et al.

3.2. Afﬁnity for carbohydrates

[19] are listed in Table 2. Therefore, it was interesting to investi-

gate these interactions by MD using the structure of FRIL deposited

Hemagglutinating activity toward native and enzyme-treated

in the database (Protein Data Bank–PDB code 1QMO). Intriguingly,

rabbit erythrocytes was fully inhibited by d-glucose (50 mM),

the immunoprecipitation assay also showed that FRIL has a high

-mannose (25 mM), N-acetyl-d-glucosamine (25 mM) and ␣-

speciﬁcity for the carbohydrates ␣␣-trehalose, trehalosamine, and

methyl- - mannopyranoside (6.25 mM), but not as much by

trimannoside [19] which justiﬁes the selection of these carbo-

100 mM d-galactose, d-xylose, N-acetyl-d-mannosamine, saccha-

hydrates as candidates for molecular docking. MD results of the

rose, and ␣- and ␤-lactose (Table 2).

interaction of FRIL with carbohydrates showed that FRIL has a

The inhibition of hemagglutinating activity by carbohydrates,

higher afﬁnity for the carbohydrate trimannoside (−135.618 MDS)

determined as minimum inhibitory concentration (MIC), was com-

(Fig. 2), followed by trehalosamine (−127.072 MDS) and ␣␣-

pared to the result obtained by Mo et al. [19], who performed

trehalose (−121.729 MDS). The MDS values of carbohydrates

corroborate the inhibition assays for carbohydrates obtained by our

group and by Mo et al. [19] (Table 2).

3.3. FRIL induces paw edema and increases vascular permeability

and hypernociception

In doses of 3, 30 and 300 ␮g/paw, FRIL evoked dose-

dependent paw edema, increasing animal paw volumes by

2- (13.59 ± 2.73 AUC), 4- (26.21 ± 3.18 AUC) and 6- (42.98 ± 2.31

AUC) fold, respectively, in relation to the control group injected

with saline (7.04 ± 1.46 AUC) (Fig. 3A). At 3 ␮g/paw, paw

edema was shown to be signiﬁcant only at the third hour

(36.25 ± 4.2 ␮L). At 30 ␮g/paw, paw edema was initiated at

the third hour (33.75 ± 3.75 ␮L), but was signiﬁcant up to the

ﬁfth hour. Finally, at 300 ␮g/paw, edema was initiated within

30 min, peaked at the ﬁfth hour (122.5 ± 5.9 ␮L), and lasted

Fig. 1. FRIL puriﬁcation by afﬁnity chromatography. (A) Chromatogram of frac-

up to 48 h (12.5 ± 3.65 ␮L) (Fig. 3B) in comparison with saline

tion 0–60% FRIL. (B) SDS-PAGE of FRIL showing ␣ and ␤ chains. Lane 1: Molecular

(3.75 ± 2.63 ␮L). The edematogenic effect of FRIL (300 ␮g/paw)

mass markers (phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin,

at the ﬁfth hour was paralleled by a signiﬁcant increase

45 kDa; carbonic anhydrase, 29 kDa; trypsin inhibitor, 20.1 kDa and ␣-lactalbumin,

± ␮

14.4 kDa); Lane 2: FRIL. in vascular permeability about 10-fold higher (8.84 0.95 g)

532 C.S. Teixeira et al. / Process Biochemistry 49 (2014) 529–534

Fig. 2. Molecular docking of FRIL. (A) The interactions of trimannoside in the carbohydrate recognition domain of FRIL structure. Yellow lines represent H-bonds with cutoff

between 2.2 and 3.2 A.˚ (B) Ligplot representation of hydrophobic and H-bond interactions of the trimannoside in the carbohydrate recognition domain of FRIL structure.

compared to saline (0.03 ± 0.26 ␮g Evans Blue/g paw wet weight) and hypernociception at the 5th h (FRIL: 63.87 ± 7.9% vs.

(Fig. 3C). FRIL + ␣-methyl-d-mannoside: 38.06 ± 7.0%), both elicited by

FRIL (300 ␮g/paw) signiﬁcantly raised the animals ﬂinch reac- FRIL. Neither saline nor ␣-methyl-d-mannoside induced edema

tion in the ﬁrst hour (76.17 ± 8.8%), third hour (76.16 ± 4.9%) and per se (Fig. 3D).

ﬁfth hour (63.87 ± 7.9%) in response to the mechanical stimulation

of Von Frey ﬁlament compared to saline (1st h: 2.30 ± 2.3%; 3rd h:

9.48 ± 3.3; 5th h: 7.12 ± 4.9%) (Fig. 4). 3.5. FRIL induces local acute inﬂammation in paw tissues

The histopathological analysis of animal paws injected with

3.4. ˛-Methyl-d-mannoside partially inhibits the edematogenic

FRIL (300 ␮g/paw), median 3 (2–3), showed, in comparison with

and hypernociceptive effects elicited by FRIL

saline median 0 (0–1), p = 0,0109 (Fig. 5A and B), a characteristically

acute inﬂammatory process that included severe mixed leukocyte

Injection of ␣-methyl-d-mannoside associated with FRIL

inﬁltrates, changes in cytoarchitecture, edema and focal areas of

(300 ␮g/paw) inhibited edema at the third hour (FRIL: 29.45 ± 4.5

hemorrhage (Fig. 5C and D).

AUC vs. FRIL + ␣-methyl-d-mannoside: 14.85 ± 3.95 AUC)

Fig. 3. FRIL induces paw edema and increases vascular permeability. Mice received s.c. saline (0.01 mL/paw), FRIL (3, 30, 300 ␮g/paw), ␣-methyl-d-mannoside (0.1 M) alone

or associated with FRIL (300 ␮g/paw). Edema was measured before (time zero) and at 1/2, 1, 2–8, 24, 48 and 72 h after induction and was expressed as the increase in paw

volume (␮L) (A) or area under curve-AUC (arbitrary units) (B, D). FRIL (300 ␮g/paw) was injected and permeability (␮g of Evans Blue/g paw wet weight) was analyzed after

5 h. Evans Blue (25 mg/kg i.v.) was injected 1 h before euthanasia for paw processing and permeability expressed as ␮g of Evans Blue/g paw wet weight (C). Mean ± S.E.M.

(n = 6). Two-way ANOVA and Bonferroni test. *p < 0.05 compared to saline; #p < 0.05 compared to FRIL (0.3 ␮g/paw) or FRIL (30 ␮g/paw).

C.S. Teixeira et al. / Process Biochemistry 49 (2014) 529–534 533

with DSSTS and contain the sequence TTTKA [24]. These results

indicate that the ␣-chains are variants of the same polypeptide. It

is well known that lectins isolated from tribe Phaseoleae undergo

post-translational processing [32]. In FRIL, such variation in band

size may result from different numbers of N-glycans, differen-

tial processing of N-glycans, or C-terminal amino acid processing

[23].

Experimental data had reported histamine release from mast

cells stimulated by Phaseoleae lectins [7]. Mast cells are inti-

mately involved in the pathophysiology of inﬂammation, and their

activation promotes the release of chemical mediators responsi-

ble for tissue changes, such as vasodilatation, increased vascular

permeability and leukocyte chemotaxis [33]. In fact, focusing on

the acute phase (0–5 h) of the inﬂammatory response elicited by

FRIL, edema, increase in vascular permeability, leukocyte inﬁltra-

tion, and hypernociception in the inﬂamed paws were evidenced.

Several inﬂammatory mediators such as histamine, serotonine,

Fig. 4. FRIL elicits hypernociception. Mice received s.c. saline (0.01 mL/paw), ␣-

bradykinin and prostaglandins are associated with these events

methyl-d-mannoside (0.1 M) alone or associated with FRIL (300 ␮g/paw). The

[34].

hypernociception was measured before and at 1, 3 and 5 h after stimulation with 6

applications of von Frey ﬁlament (0.8 g) on mouse plantar surface. Mean ± S.E.M. The inﬂammatory parameters elicited by FRIL were partly

(n = 6–8). Two-way ANOVA and Bonferroni test. *p < 0.05 compared to saline; inhibited by the injection of a solution containing the lectin asso-

#p < 0.05 compared to FRIL.

ciated with the hapten ␣-d-methyl mannoside, the most effective

monosaccharide known to inhibit lectin activity in the HA. Other

4. Discussion Phaseoleae lectins, isolated from Canavalia grandiﬂora, C. ensiformis

[11], Dioclea rostrata [35], C. brasiliensis, C. gladiata, C. maritima [10]

FRIL is a glucose/mannose-binding lectin puriﬁed from the pro- and Cymbosema roseum [11] also elicit inﬂammatory responses that

tein extract of the seeds of Dolichos lablab. Its hemagglutinating could be reversed by speciﬁc carbohydrates.

activity was ﬁrst reported by Boyd and Reguera [30]. The name In silico assays conﬁrm the results obtained by Mo et al.

FRIL was originally given based on its ability to stimulate NIH [19], in which FRIL preferentially interacts with trimannoside

−

3T3 cells transfected with Flt3, a tyrosine kinase receptor that ( 135.618 MDS) (Fig. 2) that makes up the core N-glycans cell.

plays a central role in the regulation of stem cells [31]. The elec- Thus, our study supports the hypothesis that the lectin domain

␣ d

trophoresis of FRIL showed four bands, ranging from 14 to 22 kDa and the likely glycoconjugates containing - -methyl mannoside

␣

( -chain), and another small band corresponding to a relative mass residues contribute to inﬂammatory effects of FRIL. The main N-

of 10 kDa (␤-chain) similar to those previously reported by Mo glycans composed of mannose are the high mannose-containing

et al. [19]. The amino-terminal sequencing of the ␤-chain starts and complex types involved in the molecular recognition of cells

with the sequence AQSLS, and the remaining polypeptides start [21].

Fig. 5. Photomicrography of histological sections from paw subplantar tissues. Animals received FRIL (300 ␮g/paw; s.c) or saline (50 ␮L; s.c) 5 h before euthanasia. Tissues

were collected, processed and scored. Saline (0.01 mL/paw) (A and B) – showing normal tissue and absence of edema, inﬂammatory inﬁltratration or hemorrhagic areas (HE,

100× and 200×, respectively); FRIL (300 ␮g/paw; s.c) (C and D) – showing acute inﬂammatory process, with severe leukocyte inﬁltratration (arrow) and changes in tissue

cytoarchitecture (arrow) (HE, 100× and 200×, respectively).

534 C.S. Teixeira et al. / Process Biochemistry 49 (2014) 529–534

Since FRIL presents hypernociceptive and proinﬂammatory [15] Nunes BS, Rensonnet NS, Cavada BS, Dal-Secco D, Vieira SM, Teixeira EH,

et al. Lectin extracted from Canavalia grandiﬂora seeds presents potential anti-

activities that occur with participation of the lectin domain, our

inﬂammatory and analgesic effects. Naunyn Schmiedebergs Arch Pharmacol

results suggest that FRIL could be a useful tool in the study of inﬂam- 2009;379:609–16.

mation processes and, possibly, structure/function relationships. [16] Rangel TB, Assreuy AM, Pires AF, Carvalho AU, Benevides RG, Simões RC, et al.

Crystallization and characterization of an inﬂammatory lectin puriﬁed from the

seeds of Dioclea wilsonii. Molecules 2011;16:5087–103.

Acknowledgments

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