OR IG INAL

ART ICLE

Holothuria grisea agglutinin (HGA): the firstinvertebrate lectin with anti-inflammatoryeffects

Raniere da M. Mouraa†, Karoline S. Aragaob†, Arthur A. de Meloa,Romulo F. Carneiroa, Cesar B. H. Osoriob, Patricia B. Luzb,Alexandre F. S. de Queirozc, Elizeu A. dos Santosd,Nylane M. N. de Alencarb, Benildo S. Cavadaa*aDepartment of Biochemistry and Molecular Biology, Federal University of Ceara, Campus do Pici, s/n, Bloco 907,

Fortaleza, CE, 60451-970, BrazilbDepartment of Physiology and Pharmacology, Federal University of Ceara, Cel. Nunes de Melo 1127, Fortaleza, CE,

60430-270, BrazilcDepartment of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Sen.Salgado Filho

3000, Natal, RN, 59072-970, BrazildDepartment of Biochemistry, Federal University of Rio Grande do Norte, Av. Sen. Salgado Filho 3000, Natal, RN,

59072-970, Brazil

Keywords

anti-inflammatory,

antinociceptive,

echinoderm,

Holothuria grisea,

lectin

Received 17 February 2012;

revised 30 May 2012;

accepted 22 June 2012

*Correspondence and reprints:

bscavada@ufc.br

†Both authors contributed

equally to this work.

ABSTRACT

Holothuria grisea agglutinin (HGA) is a dimeric lectin of molecular mass 228 kDa

by gel filtration with monomers of 105 kDa by SDS-PAGE. The lectin is highly

thermostable as it retains full activity for 1 h at 70 °C. Unlike other lectins purified

from marine invertebrates, the hemagglutination activity of HGA does not require

any divalent metal ions. The affinity analysis of HGA showed that only mucin was

able to inhibit the hemagglutinating activity. HGA administered intravenously was

tested in classical models of nociception and inflammation. HGA was able to inhibit

neutrophil migration into the peritoneal cavity induced by carrageenan. This

inhibitory effect was 68% at a dose of 1 mg/kg. In acetic acid-induced writhing

tests, a significant antinociceptive effect was observed by treatment with HGA (0.1;

1 or 10 mg/kg) reducing constrictions by 27, 90 and 84%, respectively. In forma-

lin tests, HGA at a dose of 10 mg/kg showed antinociceptive effect only in the

inflammatory phase (phase 2). Nevertheless, in hot-plate tests, HGA did not show

any nociceptive effect. In rota-rod and open-field tests, HGA did not alter the ani-

mals’ behavior. The treatment with HGA 10 mg/kg presented diminished myelop-

eroxidase activity activity (81.6% inhibition) and raised the circulating levels of

NO by 50.4% when compared with the carrageenan group. HGA has

demonstrated the ability to modulate the inflammatory response in models of

inflammation in vivo. HGA is the first marine invertebrate lectin that showed an

anti-inflammatory effect. This finding opens a new perspective on the potential of

lectins from the marine environment.

INTRODUCT ION

Lectins are ubiquitous sugar-binding proteins, which

possess at least one noncatalytic domain with high

specificity for carbohydrate motifs [1]. Lectins present a

wide range of activities, such as mitogenesis, toxicity in

cells, cellular adhesion, cell–cell interactions, apoptosis,splicing of RNA, tumor metastasis, agglutination of

ª 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 1

doi: 10.1111/j.1472-8206.2012.01073.x

Fund

amen

tal &

Cli

nica

l Pha

rmac

olog

y

cells, fungi and bacteria [2–6]. The biological property

of lectins in carbohydrate recognition turns those pro-

teins into powerful tools in processes of isolation and

characterization of oligosaccharides. It has become fun-

damental in investigations concerning alterations and

changes in the structure of glycans in the cellular sur-

face of malignant cells, as well as immunological stud-

ies [7]. A considerable number of marine invertebrate

lectins have already been purified and characterized,

and considerable attention is focused on its biological

recognition role [8–12]. Currently, there are strong

indications that lectin presence in marine invertebrates

is involved, mainly, in the innate defense processes

against microbial agents by complement system activa-

tion. Then, they come as ancestral molecules that

show a high degree of conservation during the evolu-

tion to superior vertebrates [13–16]. Nevertheless,

when appraised regarding the modulation of the

nonself immune system, they commonly present pro-

inflammatory activities and toxicity in cells and organ-

isms. The Table I summarizes the origin, specificity, and

biological activity from some lectins [5,17–35]. Holo-

thurians are sea animal belonging to the echinoderm

phylum with a leathery skin and an elongated body

greatly extended in the oral/aboral axis and presenting

bilateral symmetry. They are commonly known as sea

cucumber and frequently used in popular medicine by

the Asians in the treatment of arthritis, tendon and

articulation lesions, pains, muscular inflammations and

hypertension. Those animals are widely distributed

throughout the oceans and there are about 1500 spe-

cies worldwide. They are found from the intertidal zone

to the bottoms of abyssal plains. In detriment of the

great diversity of species inside the Holothuroidea class,

few lectins were isolated and characterized. The biologi-

cal properties of lectins investigated in this class pre-

sented antibiotic activity and recognition of the antigen

T [36,37], toxicity in leukemia cells [19], induction of

TNF-alpha and G-CSF [18] and hemolytic activity for

pore formation in membranes [38].

In this work, we presented the first marine inverte-

brate lectin purified from Holothuria grisea that presents

anti-inflammatory activity. Studies about this inverte-

brate lectin have been intensifying because it can be a

Table I Some lectins and their effects on the immune system.

Lectin Origin Specific sugar Biological activity

CFAL Clitoria fairchildiana [22] – Antinociceptive and

anti-inflammatory

ConGF Canavalia grandiflora [21] D-glucose/D-mannose Anti-inflammatory

Cbol Canavalia boliviana [23] D-glucose/D-mannose Antinociceptive

CRLI Cymbosema roseum [24] D-mannose Anti-inflammatory and

pro-inflammatory

BBL Bauhinia bauhinioides [20] D-galactose Pro-inflammatory

DrosL Dioclea rostrata [25] D-glucose/D-mannose Pro-inflammatory

LAL Luetzeuburgia auriculata [26] N-acetyl-D-galactosamin/

D-lactose/D-melibiose/

D-galactose

Anti-inflammatory

LSL Lonchocarpus sericeus [27] N-acetyl-glucosamine Anti-inflammatory

AMA Arum maculatum [28] Oligo-mannosidic- and

N-acetyllactosaminic-type

glycans

Pro-inflammatory

BC2L-C Burkholderia cenocepacia [29] D-mannose Pro-inflammatory

PcL Pterocladiella capillacea [30] Mucin Anti-inflammatory

CvL Cliona varians [17] D-galactose Pro-inflammatory

HCL Haliclona cratera [31] D-galactose/N-acetyl-

D-galactosamine

Cytotoxic

ACL-I Axinella corrugata [32] N-acetyl-D-glucosamine Pro-inflammatory

CaL Cinachyrella apion [33] D-lactose Cytotoxic

CVL Chaetopterus variopedatus [34] b-galactose Anti-HIV

BBL Belamvia bengalensis [35] N-acetyl-D-glucosamine/

N-acetyl-D-alactosamine

Stimulated the

T lymphocyte proliferation

(Th1)

CEL-I Cucumaria echinata [18] N-acetyl-D-glucosamine Pro-inflammatory

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

2 R.M. Moura et al.

powerful tool in the study of inflammatory modulation,

antinociceptive and antitumor effects and in the diag-

nosis of malignant cells [5,39–41].

MATER IAL AND METHODS

Material

Human erythrocytes types A, B and O were donated by

the HEMOCE Blood Bank, in Fortaleza-CE. The follow-

ing reagents were used: papain, trypsin, carrageenan

and inhibitory sugar (Sigma, St. Louis, MO, USA);

acetic acid (Grupo Quımica Brasil, Sao Paulo, SP,

Brazil.); diazepam (Roche, Sao Paulo, SP, Brazil); for-

mol (Merck, Barueri, SP, Brazil); hematoxylin (Reagen,

Parana, Brazil); heparin (Cristalia, Ponte Preta, SP,

Brazil); saline solution (Pharmace, Barbalha, CE,

Brazil); Turk’s solution (Isofar, Duque de Caxias, RJ,

Brazil); morphine sulfate (Cristalia).

Animals

Adult specimens of Holothuria grisea were collected on

the shore of St Rita beach, RN, Brazil, and Bitupita, CE,

Brazil. Specimens were collected and washed in marine

water and transported in clear marine water to the lab-

oratory where they were gutted, and the bodies were

stored at �20 °C.Adult male Wistar rats (160–220 g) and Swiss mice

(25–30 g) were obtained from the Central Animal

House of the Federal University of Ceara and housed at

25 ± °C under a 12/12 h light/dark cycle, and food

and water ad libitum. Experiments were performed

according to the Guide for the Care and Use of Labora-

tory Animals of the U.S. Department of Health and

Human Services (NIH publication no. 85-23, revised

1985) and approved by the Institutional Animal Care

and Use Committee of the Federal University of Ceara

(UFC), Fortaleza, Brazil.

Hemagglutinating assay

The activity was assayed in microtiter V plates (Nunc

Brand products, Slangerup, Denmark) according to

Debray et al. [42]. The blood cells were used native or

treated with papain or trypsin according to Benevides

et al. [43]. One hemagglutinating unit (HU) was

defined as the amount of lectin able to agglutinate and

hence precipitate the erythrocytes in suspension after

30 min. Twenty-five microlitre of the twofold serially

diluted lectin solutions and 25 lL of treated and

untreated human erythrocytes (2% v/v suspension)

were added to each well and allowed to incubate for

30 min at room temperature. The degree of hemagglu-

tinating activity was examined. The controls were set

up with saline and erythrocytes and 1 mg/mL of ConBr

solution and erythrocytes.

Purification of Holothuria grisea agglutinin (HGA)

Specimens were cut into small pieces using sharp scis-

sors. The pieces were washed in distilled water and

lyophilized. The pieces were reduced in mortar and pes-

tle and extracted (1 : 2, w/v) with 0.05 M Tris–HCl buf-fer pH 7.6, for 2 h at room temperature. After

centrifugation for 30 min at 12 000 9 g at 4 °C, thesupernatant (crude extract) was precipitated with ace-

tone at 0.5, 1.0 and 2.0 vol. These fractions (F1, F2 and

F3) were then freeze dried and submitted to assays. The

F2 fraction showed a higher level of hemagglutinating

activity for human papain-treated type A erythrocytes.

This fraction was applied to TSKgel G3000SWXL

(1.28 9 30 cm) chromatography, equilibrated with

50 mM Tris–HCl, 150 mM NaCl buffer, pH 7.6. The

active fraction was applied to HITRAP DEAE FF chro-

matography equilibrated with 50 mM Tris–HCl buffer,

pH 7.6. The retained proteins were eluted by the same

buffer in a linear NaCl gradient 0–1 M. Protein content

of all fractions was measured as described by Bradford

[44], and chromatography was monitored at 280 nm.

Carbohydrate specificity

All inhibitors tested were dissolved in 150 mM NaCl at

an initial concentration of 200 mM for monosaccharides

(D-galactose, D-glucose, D-mannose, D-fucose, D-arabi-

nose, D-N-acetyl-glucosamine and D-galactoneuraminic

acid), disaccharides (lactulose, sucrose and lactose) and

glycoprotein 5 mg/mL (mucin). An equal volume of the

lectin solution was added to 25 lL of the twofold seri-

ally diluted inhibitor solutions, and the plate was incu-

bated for 1 h at room temperature. Twenty-five

microliters of human erythrocytes (2% v/v suspension)

was added to each well and allowed to incubate for

30 min at room temperature. Results were expressed as

the minimal sugar or glycoprotein concentration

required to inhibit hemagglutinating doses of the lectin.

Effects of metal ions, temperature and pH onhemagglutinating activity

The effect of pH on the lectin activity was studied by a

hemagglutinating assay of the lectin after dialysis

against buffers of different pH levels ranging from pH

2.5 to pH 10.5 for 3 h. Fifty millimolar glycine–HClbuffer (pH 2.5), 50 mM acetate buffer (pH 4.5), 50 mM

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 3

phosphate buffer (pH 6.5), 50 mM Tris–HCl buffer (pH

8.5) and 50 mM glycine–NaOH buffer (pH 10.5) were

used. The effect of divalent metal ions on the hemagglu-

tinating activity of the lectin was assessed as follows.

The lectin was dialyzed exhaustively against 50 mM

EDTA followed by dialysis against 50 mM Tris–HCl buf-fer, pH 7.6. The hemagglutinating activity was tested in

the presence and in the absence of 100 mM Ca2+, Mg2+

and Mn2+. To study the effects of temperature on

hemagglutinating activity, lectin (0.1 mg/mL) was seri-

ally diluted, incubated at 4, 10, 20, 30, 40, 50, 60, 70,

80, 90 and 100 °C for 30 min at each temperature

and assayed after the hemagglutinating activity.

Sub-units determination, molecular massestimation

SDS polyacrylamide (12%) gel electrophoresis (SDS-

PAGE) in the absence and presence of b-mercaptoetha-

nol (0.1 M) was conducted to estimate the molecular

mass of the lectin and its sub-units by comparing with

the mobility of bands with protein molecular weight

markers (kDa): ConBr Alpha chain (25), ConBr Beta

chain (14) and ConBr Gamma chain (12). Proteins were

detected by staining with 0.1% Coomassie brilliant blue

R-250. Also, the molecular mass of HGA was estimated

by TSKgel G3000SWXL (1.28 9 30 cm) gel filtration

column calibrated with protein markers (kDa): conalbu-

min (Mr 74), ovalbumin (Mr 43), carbonic anhydrase

(Mr 29), ribonuclease (Mr 13,5) and aprotinin (Mr 6,5).

Neutrophil migration to peritoneal cavity inducedby indirect chemotactic agent (carrageenan-Cg)

Holothuria grisea agglutinin (0.1 or 1 mg/kg) diluted in

sterile saline (0.9%, NaCl) was administered intrave-

nously (i.v.; retrorbital plexus) 30 min before intraperi-

toneal (i.p.) injection of the inflammatory stimulus

(carrageenan – 500 lg/cavity/1 mL saline). The con-

trol groups received, respectively, carrageenan (i.p.) and

sterile saline (i.p. and i.v.). Rats were killed 4 h after

carrageenan injection and the peritoneal cavity

was washed with 10 mL of saline containing heparin

(5 UI/mL). The peritoneal fluid was collected, and total

and differential leukocyte counts were carried out

according to Souza & Ferreira [45]. Results were

expressed as mean ± EPM, the number of cells 9 103/

mL peritoneal fluid.

Acetic acid-induced writhing test

The experiment was performed according to Koster

et al. [46], with some modifications. Mice were treated

with HGA (0.1; 1 or 10 mg/kg, i.v.), 30 min before

i.p. administration of 0.6% acetic acid (10 mL/kg

body weight). Ten minutes after administration of the

acid, the number of constrictions was counted for

20 min. The writhing response consists of a contrac-

tion of the abdominal muscle together with a stretch-

ing of the hind limbs. Results were expressed as

mean ± EPM, the number of writhing/20 min.

Formalin test

Run according to Hunskaar and Hole [47], mice were

treated with HGA (1 or 10 mg/kg, i.v) 30 min before

receiving 20 lL of formalin 1.5% s.c. (v/v in distilled

water) in right hind paw. Soon after administration of

formalin, the time (in seconds) that animals spent

licking the injected paws was counted for 5 min

(phase 1, neurogenic), and after 15 min this was

observed again for a further 5 min (phase 2, inflam-

matory). The control groups received, respectively,

morphine (5 mg/kg s.c., reference drug) and saline

(i.v.) 30 min before formalin injection. Results were

expressed as mean ± EPM, the licking time in

seconds.

Hot-plate test

Mice were placed on a plate heated to 55 °C (±1 °C),and a measurement was made of the time they

remained on the board until proof of stereotyped

behavior in reaction to pain (licking or jumping). The

basal time (0 time) was measured before treatment,

and the animals that did not respond by the end of

20 s were eliminated from the test. Soon after, treat-

ment was undertaken with HGA (1 or 10 mg/kg, i.v.),

morphine (5 mg/kg, s.c.) or saline (i.v.) and the reac-

tion times were recorded in time intervals of 30, 60,

90 and 120 min after treatment, with a cut-off time of

45 s to avoid animal paw lesion. Results were

expressed as mean ± EPM, the reaction time in seconds

[48].

Rota-rod test

In an attempt to demonstrate some possible muscle

relaxing or sedative effect, the rota-rod test was run,

which consists of a bar 2.5 cm in diameter rotating at

4 rpm [49]. Mice were previously selected 1 day in

advance and those that remained <2 min on the bar

were excluded from the test. The animals were treated

with HGA (1 or 10 mg/kg, i.v.), diazepam (5 mg/kg,

i.p., reference drug) or saline (i.v.). Thirty minutes

later, animals were placed individually on the bar and

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

4 R.M. Moura et al.

the permanence time was registered during 2 min.

Results were expressed as mean ± EPM, the perma-

nence time (seconds) of the animal on the bar.

Open-field test

To verify motor activity, mice were treated with HGA

(1 or 10 mg/kg, i.v.), diazepam (5 mg/kg, i.p., refer-

ence drug) or saline (i.v.) 30 min before open-field test-

ing. Animals were placed wandering freely in an open

field (30 9 30 9 15 cm) divided in nine squares of

equal area, during 1 min for ambiance. Then, during

the next 4 min, the number of their crossings from one

marked area in the field to another was observed and

counted [50].

Determination of myeloperoxidase activity (MPO)

Myeloperoxidase activity, a kinetic-colorimetric assay,

was used to measure neutrophil accumulation in the

mice’s plantar tissues. Mice were treated with HGA

(10 mg/kg, i.v.) or saline (i.v.), 30 min later they

received 300 lg/paw of carrageenan in the right hind

paw, 4 h later they were killed, and approximately

0.25 cm2 of plantar tissue was removed and homogen-

ated in hexadecyltrimethylammonium bromide (HTAB/

50 mM K2PO4 buffer pH 6.0) using a polytron homoge-

nizer (two cycles of 30 s at maximum speed). After

centrifugation at 10 621 g. for 5 min at 4 °C, super-natant fractions were assayed for MPO activity, as an

index of cellular migration, using the method described

by Bradley et al. [51]. To prepare the solution for anal-

ysis, 7 lL of supernatant was mixed with 200 lL of

50 mM phosphate buffer, pH 6.0, containing

0.167 mg/mL O-dianisidine dihydrochloride and

0.0005% hydrogen peroxide. MPO activity was mea-

sured by the absorbance of the solution at 450 nm

(Asys Hitech, Expert Plus, Eugendorf, Austria.), taking

three readings at 1-min intervals. Calculation of units

of MPO was carried out considering that 1 U

MPO = 1 mmol H2O2 split and 1 mmol H2O2 gives a

change in absorbance of 1.13 9 10�2 (change in

absorbance nm/min).

Nitric oxide assay

Mice were treated with HGA (10 mg/kg, i.v.) or saline

(i.v.), 30 min later they received 300 lg/paw of carra-

geenan in the right hind paw and 4 h later they were

killed. Serum was incubated on a microplate with

nitrate reductase (0.016 U/well) for 12 h to convert

NO3 to NO2. Nitric oxide (NO) production was deter-

mined using the Griess method by measuring nitrite

concentrations in an ELISA plate reader at 540 nm, and

the results were expressed as micromoles of nitrite [52].

RESULTS

HGA purification

Crude soluble protein extract (CE) obtained from sea

cucumbers was initially precipitated at 0.5, 1.0 and

2.0 vol with acetone and three protein fractions were

obtained (F1, F2 and F3). The F2 protein fraction

showed strong hemagglutinating activity for papain-

treated type A erythrocytes, while the other fractions

exhibited low activity (F1) and hemolysis (F3). The F2

fraction was then applied to TSKgel G3000SWXL col-

umn (0.15 m 9 30 cm) gel filtration chromatography

and the peaks were submitted to a hemagglutinating test

(Figure 1). The active fraction (PI TSK) was collected

and applied to HITRAP DEAE FF chromatography. The

adsorbed peak (PII DEAE) showed hemagglutinating

activity (Figure 2). This process resulted in a purification

of 7.5-fold with a 37.5% recovery (Table II). The hemag-

glutinating activity of the CE could not be determined by

the presence of hemolysis.

Lectin specificity

As shown in Table III, the effects of monosaccharides,

oligosaccharides and glycoprotein on HGA hemaggluti-

nation activity were examined. Hemagglutinating

activity of HGA toward papain-treated type A erythro-

cytes was only inhibited by mucin.

Physical and chemical analysis of the HGA

The lectin purification procedure from the sea cucum-

ber Holothuria grisea was observed with SDS-PAGE

TSKGel HGA 20 07 09:1_UV2_280 nm

0

50

100

150

200

250

300

mAU

0.0 5.0 10.0 15.0 20.0 mL

3

21

Figure 1 Holothuria grisea F2 elution profile on TSKGel

G3000SWXL.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 5

(Figure 3). Electrophoretic analysis of HGA in the

absence of a reducing agent (b-mercaptoethanol)

showed one protein band with molecular mass of

approximately 105.7 kDa. The relative molecular mass

of the native lectin was estimated at 228 kDa by gel fil-

tration on a calibrated TSKGel G3000SWXL column

(Figure 4). The study of the temperature effect on HGA

Table II Purification processes of Holothuria grisea agglutinin

lectin.

Purification

fractions TP (mg) Titer HU/mg

Total

activity Purification

Recovery

%

C.E. 271.2 nd nd nd nd nd

F2 38.4 512 853.3 32 768 1.0 100

PI TSK 6.6 512 2031.7 13 312 2.4 40.63

PII DEAE 1.9 1024 6400 12 288 7.5 37.5

TP, Total protein; HU, Hemagglutination Unit; nd, not determined.

Table III Inhibition of hemagglutinating activity of Holothuria

grisea agglutinin.

Inhibitor compounds MIC

Glucose 0.1 M NI

Mannose 0.1 M NI

Galactose 0.1 M NI

Fucose 0.1 M NI

N-Acetyl Glucosamine 0.1 M NI

Galactoneuraminic acid 0.1 M NI

Arabinose NI

Lactulose NI

Lactose 0.1 M NI

Sucrose 0.1 M NI

Mucin 5 mg /mL 0.65 mg/mL

Fucoidan 5 mg /mL NI

Carrageenan 5 mg /mL NI

MIC, minimum inhibitory concentration; NI, sugar not inhibitory until a

concentration of 200 mM.

Figure 3 SDS-PAGE final steps of Holothuria grisea agglutinin

purification. (M) Molecular weight markers (kDa): BSA (67)

ConBr Alpha chain (25), ConBr Beta chain (14), ConBr Gamma

chain (12). Proteins were stained with Coomassie blue. 1 = P1

TSK, 2 = PI DEAE, 3 = PII DEAE.

Raniere DEAE HGA 02 26 05 10:1_UV1_280 nmRaniere DEAE HGA 02 26 05 10:1_Conc

0

100

200

300

400

500

mAU

0

20

40

60

80

100%B

0.0 5.0 10.0 15.0 20.0 mL

21

Figure 2 HITRAP DEAE FF profile lectin-active fraction 1 from

TSKGel G3000SWXL column was applied to column yielding

lectin-active fraction 2. Dotted line denotes the linear gradient

(0–1 M) of NaCl.

0.0 0.5 1.0 1.5 2.03.5

4.0

4.5

5.0

5.5

6.0

ConalbuminOvoalbumin

Carbonic anhydrase Ribonuclease

Aprotinin

HGA

y = −0.9750X + 5.5279

R2 = 0.9939

KAV

Log(

WT)

Figure 4 Determination of molecular weight of Holothuria grisea

agglutinin by gel filtration on TSKGel G3000SWXL

chromatography. Proteins were applied in the column

equilibrated with 50 mM Tris–HCl 150 mM NaCl buffer, pH 7.0.

The eluted protein fractions were monitored at 280 nm. Protein

molecular weight standards were: conalbumin (74 kDa),

ovalbumin (43 kDa), carbonic anhydrase (29 kDa) ribonuclease

(13,5 kDa) and aprotinin 6,5 kDa. Phase distribution coefficient

(Kav).

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

6 R.M. Moura et al.

showed that the lectin was stable at 70 °C and had

abolished its activity at 100 °C (Figure 5). The hemag-

glutinating activity of HGA was not metal ion

dependent.

Neutrophil migration in rats induced byintraperitoneal injection of carrageenan

Figure 6 shows that 4 h after administration of inflam-

matory stimulus (Cg, 500 lg/cavity, i.p), a significant

increase (P < 0.05) was observed in the neutrophil

migration in the peritoneal cavity compared with the

saline group, injected only with saline i.p. and i.v.

(428.7 ± 172.2 to 8832 ± 799.9 9 103 cells/mL).

Treatment of rats with HGA at dose 1 mg/kg, i.v.,

30 min before Cg caused significantly decreased neu-

trophil migration when compared with Cg group

(2849 ± 828.4), 68% inhibition. HGA at dose of

0.1 mg/kg did not significantly decrease neutrophil

migration induced by Cg (i.p.).

Acetic acid-induced writhing test

Treatment with HGA i.v. 30 min before the i.p. injec-

tion of 0.6% acetic acid showed a significant antinoci-

ceptive effect inhibiting the number of abdominal

writhing episodes evoked by the stimulus when com-

pared with mice injected with acetic acid (i.p.) plus sal-

ine (i.v.) (Table IV). HGA (0.1; 1 or 10 mg/kg) reduced

the number of constrictions by 27, 90 and 88.4%,

respectively. The positive control, treated with the ref-

erence drug morphine (5 mg/kg, s.c.), also manifested

strong analgesic effect (by about 99%).

Formalin test

The treatment with HGA (1 or 10 mg/kg, i.v.), 30 min

before formalin (1.5%, s.c., in right hind paw), showed

a significant antinociceptive effect, reducing the licking

time only in the inflammatory phase (phase 2) of the

test. The percentage reductions were 69.9% compared

with the control group, injected only with saline. HGA

at a dose of 1 mg/kg did not produce a significant

antinociceptive effect in any phase. As expected,

morphine significantly reduced the formalin response

in both phases, by 60.0 and 98.2%, respectively. This

result confirms the activity of opiates in both phases as

shown in previous studies [53,54] (Table V).

0 20 40 60 80 1000

20

40

60

80

100

Temperature °C

% H

.U.

Figure 5 The temperature effects on the hemagglutination

activity of Holothuria grisea agglutinin.

0

5000

10 000

15 000

– 10.1

*

#

HGA (mg/Kg, i.v.)

Cg (500 µg/cavity)

Sal

Neu

trop

hils

x 1

03/m

L

Figure 6 Effect of Holothuria grisea agglutinin (HGA) on

neutrophil migration induced by carrageenan (Cg). Neutrophil

migration was induced by i.p. injection of Cg (500 lg/cav/1 mL

saline) and evaluated 4 h later. Rats were pretreated 30 min

before Cg with HGA (0.1 or 1 mg/kg, i.v.) or saline (i.v.; i.p.).

Results are shown as the mean ± SEM. (n = 5). *P < 0.001

when compared with the saline (Sal) group. #P < 0.001 when

compared with the Cg group. ANOVA followed by Bonferroni’s test.

Table IV Effect of Holothuria grisea agglutinin (HGA) in the

abdominal constrictions induced by 0.6% acetic acid. Mice were

pretreated 30 min before acetic acid with saline (i.v.), morphine

(reference drug, diluted in saline – 0.9% NaCl) or HGA (diluted in

saline – 0.9% NaCl). Results are shown as the mean ± SEM

(n = 8).

Experimental

groups

Number of abdominal

constrictions (20 min) % Inhibition

Saline 37 ± 2.38

Morphine (5 mg / kg. s.c.) 0.1 ± 0.12** 99.7

HGA 0.1 (mg / kg. i.v.) 27 ± 3.8* 27.0

HGA 1 (mg / kg. i.v.) 3.7 ± 1.47** 90.0

HGA 10 (mg / kg. i.v.) 4.3 ± 2.01** 88.4

*P < 0.01 and **P < 0.001 when compared with the saline group (Saline).

ANOVA followed by Student Neuman-Keuls.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 7

Hot-plate test

Treatment with HGA (1 or 10 mg/kg, i.v.) did not

show any antinociceptive effect in the hot-plate test.

Lectin in doses of 1 or 10 mg/kg i.v. did not signifi-

cantly delay (P < 0.05) the time of reaction in the hot-

plate test at any of the analyzed time points when

compared with the control group, injected only with

saline. The administration of the reference drug mor-

phine showed antinociception (Table VI), with a delay

in the reaction time of 41 ± 2.6, 36.8 ± 4.7 and

27.1 ± 4.1 s compared with saline 16.3 ± 1.9,

13.6 ± 2.1 and 10.8 ± 1.6 s at the 30, 60 and

90 min marks, respectively.

Rota-rod and open-field tests

Treatment of animals with HGA (1 or 10 mg/kg, i.v.)

did not alter the number of crossings, rearing or immo-

bility in the open-field test compared with control

group, inject only saline (Figure 7). Similarly, HGA, at

any dose tested, did not alter the behavior responses in

the rota-rod test compared with control group, inject

only saline (Figure 8). Diazepam (5 mg/kg, i.p.)

treatment, reference drug, significantly reduced

(P < 0.05) permanence time in bar (30.2 ± 4.5 s) and

number of fields crossing/4 min (5.4 ± 1.6 campos

explorados/4 min) compared with saline groups.

Myeloperoxidase activity (MPO)

The animals treated with HGA 10 mg/kg i.v. presented

diminished MPO activity (81.6% inhibition) when com-

pared with the group who received intraplantar admin-

istration of carrageenan (300 lg/paw). This means

that HGA inhibited the migration of neutrophils to the

inflammation site (Figure 9).

Nitric oxide assay

Holothuria grisea agglutinin 10 mg/kg i.v. administered

30 min before carrageenan (300 lg/paw) raised the

circulating levels of NO by 50.4% when compared

with the group that received only the phlogistic agent

Table V Effect of Holothuria grisea agglutinin (HGA) in the paw

licking induced by formalin 1.5%. Mice were pretreated 30 min

before formalin (formaldehyde diluted in saline – 0.9% NaCl) with

saline (i.v.), morphine (reference drug, diluted in saline – 0.9%

NaCl) or HGA (diluted in saline – 0.9% NaCl).Results are shown

as the mean ± SEM (n = 8).

Experimental

groups

Neurogenic phase Inflammatory phase

Phase 1

(seg)

Inhibition

(%)

Phase 2

(seg)

Inhibition

(%)

Saline 60.0 ± 2.8 46.5 ± 5.6 –

Morphine

(5 mg / kg. s.c.)

24.0 ± 1.4** 60.0 0.86 ± 0.6** 98.2

HGA

(1 mg / kg. i.v.)

52.2 ± 5.5 13.0 46.4 ± 9.2 –

HGA

(10 mg / kg. i.v.)

52.5 ± 3.4 12.7 14.4 ± 8.6* 69.9

*P < 0.01 and **P < 0.001 when compared with saline group (saline).

ANOVA followed by Student Neuman-Keuls.

Table VI Effect of Holothuria grisea agglutinin (HGA) in the hot plate test. Measurements were performed before (basal time) and 30, 60,

90 and 120 min after treatment. Mice were pretreated 30 min before testing with saline (i.v.), morphine (reference drug) or HGA.

Results are shown as the mean ± SEM (n = 8).

Experimental groups

Reaction time (s)

Basal 30 min 60 min 90 min 120 min

Saline 17.7 ± 1.9 16.3 ± 1.9 13.6 ± 2.1 10.8 ± 1.6 12.1 ± 2.8

Morphine (5 mg / kg. s.c.) 13.6 ± 1.4 41 ± 2.6** 36.8 ± 4.7* 27.1 ± 4.1** 13.5 ± 0.8

HGA (1 mg / kg. i.v.) 15.1 ± 1.1 18.8 ± 2.4 13.9 ± 2.3 16.2 ± 1.7 15.7 ± 3.2

HGA(10 mg / kg. i.v.) 16.1 ± 1.2 12.9 ± 1.5 15.4 ± 1.6 9.6 ± 0.6 13.5 ± 2.9

*P < 0.01 and **P < 0.001 when compared with the sterile saline group (Saline). ANOVA followed by Student Neuman-Keuls.

0

20

40

60

Tota

l fie

lds

oper

ated

/4 m

in

Sal Diazepam(5 mg/kg, i.p.)

1 10HGA (mg/kg, i.v.)

*

Figure 7 Effect of Holothuria grisea agglutinin (HGA) in the open-

field test. Mice were pretreated 30 min before test with saline

(i.v.), diazepam (5 mg/kg, i.p., diluted in saline – 0.9% NaCl) or

HGA (diluted in saline – 0.9% NaCl). Results are shown as the

mean ± SEM. (n = 8). *P < 0.001 when compared with the

saline group (Sal). ANOVA followed by Bonferroni’s test.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

8 R.M. Moura et al.

(carrageenan). This result means the lectin caused an

increased production of NO, once we can infer the

assayed NO2 and NO3 are its direct metabolites (Fig-

ure 10).

DISCUSS ION

Lectins have been isolated from various marine inverte-

brates, and great attention is being given regarding

their application as tools in biotechnology. Invertebrate

lectins make an important contribution to innate

immune protection and work along with epithelial

barriers, cellular defenses such as phagocytosis, and

pattern-recognition receptors that trigger pro-inflam-

matory signaling cascades [55]. In this work, we puri-

fied a novel lectin from gutted bodies of Holothuria

grisea. HGA demonstrated the ability to agglutinate

human erythrocytes with preference for type A treated

with papain. The lectin is present in native form com-

posed of two subunits not covalently linked with

molecular mass of 228 kDa, its monomer containing

105 kDa and was visualized by SDS-PAGE. Marine lec-

tins are generally identified by their metal ion require-

ment for their hemagglutination activity. However,

HGA did not present the metal ion dependence, simi-

larly to Holothuria scabra (HSL) [36,37]. Its inhibition

of mucin coupled with the absence of inhibition by

monosaccharides suggests an affinity for O-glycans

motifs such as HSL and Holothuria atra lectin

[36,37,56]. Further analysis will be conducted aiming

to characterize in detail the affinity profile of the HGA.

The development of biotechnological tools for therapeu-

tic applications becomes of great interest.

The carrageenan-induced peritonitis is an experimen-

tal model of acute inflammation well characterized and

largely employed to test new anti-inflammatory thera-

pies, and consists of the quantification and correlation

of cellular migration of inflammatory exudate [43].

This study demonstrated that the lectin isolated from

the gutted body of the marine invertebrate Holothuria

grisea administered i.v. at dose of 1 mg/kg inhibited

0

50

100

150R

esid

ence

tim

ein

the

bar (

s)

Sal Diazepam(5 mg/kg, i.p.)

1 10HGA (mg/kg, i.v.)

*

Figure 8 Effect of Holothuria grisea agglutinin (HGA) in the rota-

rod. Mice were pretreated 30 min before test with saline (i.v.),

diazepam (5 mg/kg, i.p., diluted in saline – 0.9% NaCl) or HGA

(diluted in saline – 0.9% NaCl). Results are shown as the

mean ± SEM. (n = 8). *P < 0.001 when compared with the

saline group (Sal). ANOVA followed by Bonferroni’s test.

0

2

4

6

8

*

#

MPO

U/m

g

Sal – 10HGA (mg/kg, i.v.)

Cg (300 µg/paw)

Figure 9 Effect of pretreatment with Holothuria grisea agglutinin

(HGA) lectin on the influx neutrophils into the hind paw of mice

after Cg (300 lg/paw/50 lL saline) administration. Saline (i.v.)

or HGA (10 mg/kg, i.v.) was injected and 30 min later Cg was

injected into the hind paws. Myeloperoxidase activity in the hind

paw was used as an index of neutrophil influx, and it was

measured 4 h after Cg. Results are shown as the mean ± SEM.

(n = 6). *P < 0.001 when compared with the saline group (Sal).

#P < 0.01 when compared with the Cg group. ANOVA followed by

Bonferroni’s test.

0

10

20

30

40

*#

Sal _ 10HGA (mg/kg, i.v.)

Cg (300 µg/paw)

NO

2– (µ

M)

Figure 10 Effect of pretreatment with Holothuria grisea agglutinin

(HGA) lectin on serum levels of nitric oxide (NO3/NO2) when Cg

(300 lg/paw/50 lL saline) was injected in the paw. Saline (i.v.)

or HGA (10 mg/kg, i.v.) was injected, and 30 min later, Cg was

injected into the hind paws. Levels of nitric oxide were measured

3 h after Cg administration. Results are shown as the

mean ± SEM. (n = 6). *P < 0.05 when compared with the saline

group (Sal). #P < 0.05 when compared with the Cg group. ANOVA

followed by Bonferroni’s test.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 9

inflammatory response, reducing the number of

neutrophils by 68% in the peritoneal cavity of animals

stimulated with carrageenan. Previous studies have

shown the ability of some lectins to reduce the rolling

and adhesion of neutrophils on the endothelium, possi-

bly by blocking interactions of adhesion molecules pres-

ent on neutrophils (L-selectins) and endothelial cells (E-

and P-selectins) [26,27,57,58]. As selectins are essen-

tial for neutrophil migration in inflammatory processes,

it was postulated that HGA could inhibit neutrophil

recruitment into inflamed tissues by competitive block-

age with a common selectin carbohydrate ligand. The

acetic acid-induced writhing test is described as a typi-

cal model of inflammatory pain. Local irritation pro-

moted by acetic acid induces the release of various

endogenous mediators such as bradykinin, prostaglan-

dins, substance P and cytokines (TNF-a, IL-1ß and IL-8)

that stimulate afferent nociceptive neurons [54,59]. This

test is sensitive to different substances with the most var-

ied mechanisms of action, via central and/or peripheral

analgesic action [46]. HGA lectin, in tested doses, signifi-

cantly inhibited the number of abdominal writhes pro-

duced in acetic acid testing, suggesting that this effect

may be related with the inhibition of some of the sub-

stances released by the chemical stimulus, or even by a

decreasing number of leukocytes in the peritoneal cavity.

The formalin test is widely used for elucidation of

mechanisms of pain and analgesia [60]. In this test,

two distinct phases of nociception occur. The neuro-

genic phase (phase 1) is characterized by direct chemi-

cal stimulation of nociceptors, mainly the C fibers,

whereas the late phase (phase 2), called inflammatory,

is related to a combination of inflammatory reaction in

peripheral tissue and functional changes in the dorsal

horn of the spinal cord [60]. The release of various

inflammatory mediators such as excitatory amino acids,

neuropeptides, PGE2, cytokines and nitric oxide occurs

in the second phase [61–63]. HGA significantly inhib-

ited only the second phase when compared with the

saline group, suggesting an antinociceptive effect only

related to inflammatory pain. Previous studies have

shown that the migration of neutrophils to the inflam-

matory focus is directly related to the painful process

[64–68]. Ribeiro et al. [69] investigated the role of resi-

dent leukocytes in the peritoneal cavity during induc-

tion caused by nociceptive acetic acid, suggesting that

the induction promoted by this nociceptive chemical

stimulation occurs through a mechanism dependent on

the presence of polymorphonuclear cells and also of

proinflammatory cytokines (TNF-a and IL-1ß). Accord-

ing to a study, Cunha et al. [70], the migration of neu-

trophils to the inflamed site is a key step for the release

of proinflammatory cytokines (TNF-a and IL-1b), and

also of mediators that act directly on nociceptors such

as PGE2 and sympathetic amines. Some lectins have

shown antinociceptive activity-dependent decrease in

neutrophil migration [27,71,72]. The antinociceptive

activity of the HGA lectin in acetic acid-induced writh-

ing and formalin tests may be related to the ability of

this protein to reduce the migration of neutrophils to

the site of injury; these data correlated with results

obtained in the peritonitis model and confirmed with

the result of myeloperoxidase (MPO).

The hot-plate test involves a specific central

response, which involves spinal and supraspinal path-

ways [73]. The morphine group caused a significant

increase in time with the animals on the heated plate

when compared with the saline group. The HGA did

not show significant activity when compared with the

morphine group, suggesting that the lectin does not

show a central antinociceptive effect, and confirming

what was observed in the formalin test (phase 1). The

depressing action of several drugs on the central ner-

vous system and muscular system can generate reduc-

tion of motor coordination in animals, as well as in the

expression of nociceptive behavior [74]. Locomotor

impairment related to the administration of certain

substances reflects the state of prolonged immobility

that may interfere with the animal’s behavior. The

rota-rod and open-field tests were conducted, respec-

tively, to evaluate a possible activity of the lecting HGA

on the animals’ motor system. The results from these

experiments demonstrated that the lectin HGA at doses

of 1 and 10 mg/kg do not interfere with locomotor

activity of mice in the rota-rod test and there was no

motor impairment when the animals were placed in

the open field. These data suggest that the behaviors

assessed in models of nociception performed in this

work were not compromised by possible nonspecific

activity of the lectin HGA on the central nervous sys-

tem. The results shown indicate that there was no sig-

nificant change when compared with diazepam

(central nervous system depressor). These data suggest

that the HGA lectin does not interfere in the reaction

of the animal through chemical (acetic acid 0.6 and

1.5% formalin) and thermal (hot plate) stimuli.

Myeloperoxidase is a peroxidase abundantly expre-

ssed in neutrophils. MPO activity in the tissue is a

direct marker of the infiltration of these cells. Animals

that were injected only with carrageenan, a known

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

10 R.M. Moura et al.

inflammatory agent that causes edema and neutrophil

infiltration, showed high MPO activity in homogenates

analyzed. Previously, treatment with HGA showed an

inhibitory effect on the migration of these cells to the

inflammatory focus, confirming the peritonitis experi-

ment results. This effect can also explain the antinoci-

ceptive action of HGA as previously explained. This

impairment of migration may be related to increased

circulation production of nitric oxide resulting from

treatment with HGA, as it has been shown that NO

down-regulates the expression of adhesion molecules in

the vascular endothelium, thereby decreasing neutro-

phil traffic into the inflamed area [65–68]. Further-

more, the administration of nitric oxide donors reduces

leukocyte infiltration in different models of inflamma-

tion [69,70]. Other studies show the relationship

between NO and pain: it reduces hyperalgesia by acti-

vation of the L-arginine/NO/cGMP pathway, causing

direct blockade of acute and persistent hypernocicep-

tion by opening K+ ATP channels. This mechanism

has been demonstrated for some analgesic drugs, such

as peripherally acting opioids and dipyrone [71].

This work demonstrated that the lectin of HGA has

anti-inflammatory activity on carrageenan-induced

peritonitis and an important antinociceptive activity in

classic models of nociception. Thus, from the more

detailed understanding of these mechanisms, we could

suggest a new pharmacological tool for processes of

inflammation and nociception.

ACKNOWLEDGEMENTS

This work received financial support from the Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico –CNPq (Brazil).

REFERENCES

1 Peumans W.J., Van Damme E.J. Lectins as plant defense

proteins. Plant Physiol. (1995) 109 347–352.

2 Akahani S., Nangia-Makker P., Inohara H., Kim H.R., Raz A.

Galectin-3: a novel antiapoptotic molecule with a functional

BH1 (NWGR) domain of Bcl-2 family. Cancer Res. (1997) 57

5272–5276.

3 Danguy A., Camby I., Kiss R. Galectins and cancer. Biochim.

Biophys. Acta (2002) 1572 285–293.

4 Nguyen J.T., Evans D.P., Galvan M., Pace K.E., Leitenberg D.,

Bui T.N., et al. CD45 modulates galectin-1-induced T cell

death: regulation by expression of core 2 O-glycans. J.

Immunol. (2012) 167 5697–5707.

5 Queiroz A.F., Silva R.A., Moura R.M., Dreyfuss J.L., Paredes-

Gamero E.J., Souza A.C., et al. Growth inhibitory activity of a

novel lectin from Cliona varians against K562 human

erythroleukemia cells. Cancer Chemother. Pharmacol. (2009)

63 1023–1033.

6 Sales M.P., Gerhardt I.R., Grossi-De-Sa M.F., Xavier-Filho J. Do

legume storage proteins play a role in defending seeds against

bruchids? Plant Physiol. (2000) 124 515–522.

7 Turner M.W. The role of mannose-binding lectin in health

and disease. Mol. Immunol. (2003) 40 423–429.

8 Alpuche J., Pereyra A., Mendoza-Hernandez G., Agundis C.,

Rosas C., Zenteno E. Purification and partial characterization

of an agglutinin from Octopus maya serum. Comp. Biochem.

Physiol. B Biochem. Mol. Biol. (2010) 156 1–5.

9 Bulgakov A.A., Nazarenko E.L., Petrova I.Y., Eliseikina M.G.,

Vakhrusheva N.M., Zubkov V.A. Isolation and properties of a

mannan-binding lectin from the coelomic fluid of the

holothurian Cucumaria japonica. Biochemistry (Mosc). (2000)

65 933–939.

10 Belogortseva N., Molchanova V., Glazunov V., Evtushenko E.,

Luk’yanov P. N-Acetyl-D-glucosamine-specific lectin from the

ascidian Didemnum ternatanum. Biochim. Biophys. Acta

(1998) 1380 249–256.

11 Mathieu S.V., Aragao K.S., Imberty A., Varrot A. Discoidin I

from Dictyostelium discoideum and Interactions with

oligosaccharides: specificity, affinity, crystal structures, and

comparison with discoidin II. J. Mol. Biol. (2010) 400 540–

554.

12 Moura R.M., Queiroz A.F., Fook J.M., Dias A.S., Monteiro N.K.,

Ribeiro J.K., et al. CvL, a lectin from the marine sponge Cliona

varians: isolation, characterization and its effects on pathogenic

bacteria and Leishmania promastigotes. Comp. Biochem.

Physiol. A Mol. Integr. Physiol. (2006) 145 517–523.

13 Hatakeyama T., Suenaga T., Eto S., Niidome T., Aoyagi H.

Antibacterial activity of peptides derived from the C-terminal

region of a hemolytic lectin, CEL-III, from the marine

invertebrate Cucumaria echinata. J. Biochem. (2004) 135

65–70.

14 Sekine H., Kenjo A., Azumi K., Ohi G., Takahashi M.,

Kasukawa R., et al. An ancient lectin-dependent complement

system in an ascidian: novel lectin isolated from the plasma of

the solitary ascidian, Halocynthia roretzi. J. Immunol. (2001)

167 4504–4510.

15 Skjoedt M.O., Palarasah Y., Rasmussen K., Vitved L.,

Salomonsen J., Kliem A., et al. Two mannose-binding lectin

homologues and an MBL-associated serine protease are

expressed in the gut epithelia of the urochordate species Ciona

intestinalis. Dev. Comp. Immunol. (2010) 34 59–68.

16 Wang X.W., Xu W.T., Zhang X.W., Zhao X.F., Yu X.Q.,

Wang J.X. A C-type lectin is involved in the innate immune

response of Chinese white shrimp. Fish Shellfish Immunol.

(2009) 27 556–562.

17 Queiroz A.F., Moura R.M., Ribeiro J.K., Lyra I.L., Cunha D.C.,

Santos E.A., et al. Pro-inflammatory effect in mice of CvL, a

lectin from the marine sponge Cliona varians. Comp. Biochem.

Physiol. C Toxicol. Pharmacol. (2008) 147 216–221.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 11

18 Yamanishi T., Yamamoto Y., Hatakeyama T., Yamaguchi K.,

Oda T. CEL-I, an invertebrate N-acetylgalactosamine-specific

C-type lectin, induces TNF-alpha and G-CSF production by

mouse macrophage cell line RAW264.7 cells. J. Biochem.

(2007) 142 587–595.

19 Sallay I., Moriwaki S., Nakamura O., Yasuda S., Kimura M.,

Yamasaki N., et al. Interaction of the hemolytic lectin, CEL-

III, with cultured human leukemic cell lines. J. Hematother.

Stem Cell Res. (2000) 9 877–883.

20 Silva H.C., Bari A.U., Pereira-Jenior F.N., Simoes R.C.,

Barroso-Neto I.L., Nobre C.B., et al. Purification and partial

characterization of a new pro-inflammatory lectin from

Bauhinia bauhinioides Mart (Caesalpinoideae) seeds. Protein

Pept. Lett. (2011) 18 396–402.

21 Simoes R.C., Rocha B.A., Bezerra M.J., Barroso-Neto I.L.,

Pereira-Junior F.N., da Mata Moura R., et al. 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. (2012) 26 811–818.

22 Leite J.F., Assreuy A.M., Mota M.R., Bringel P.H., Lacerda R.R.,

Gomes Vde M., et al. Antinociceptive and anti-inflammatory

effects of a lectin-like substance from Clitoria fairchildiana R.

Howard seeds. Molecules (2012) 17 3277–3290.

23 Figueiredo J.G., da Silveira Bitencourt F., Beserra I.G., Teixeira C.

S., Luz P.B., Bezerra E.H., et al. Antinociceptive activity and

toxicology of the lectin fromCanavalia boliviana seeds inmice.

Naunyn Schmiedebergs Arch Pharmacol. (2009)380 407–414.

24 Rocha B.A., Delatorre P., Oliveira T.M., Benevides R.G., Pires

A.F., Sousa A.A., et al. Structural basis for both pro- and

anti-inflammatory response induced by mannose-specific

legume lectin from Cymbosema roseum. Biochimie (2011) 93

806–816.

25 Figueiredo J.G., Bitencourt F.S., Mota M.R., Silvestre P.P.,

Aguiar C.N., Benevides R.G., et al. Pharmacological analysis

of the neutrophil migration induced by D. rostrata lectin:

involvement of cytokines and nitric oxide. Toxicon (2009) 54

736–744.

26 Alencar N.M., Oliveira R.S., Figueiredo J.G., Cavalcante I.J.,

Matos M.P., Cunha F.Q., et al. 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. (2010) 59 245–254.

27 Napimoga M.H., Cavada B.S., Alencar N.M., Mota M.L.,

Bittencourt F.S., Alves-Filho J.C., et al. Lonchocarpus sericeus

lectin decreases leukocyte migration and mechanical

hypernociception by inhibiting cytokine and chemokines

production. Int. Immunopharmacol. (2007) 7 824–835.

28 Alencar V.B., Alencar N.M., Assreuy A.M., Mota M.L., Brito

G.A., Aragao K.S., et al. Pro-inflammatory effect of Arum

maculatum lectin and role of resident cells. Int. J. Biochem.

Cell Biol. (2005) 37 1805–1814.

29 Sulak O., Cioci G., Lameignere E., Balloy V., Round A.,

Gutsche I., et al. Burkholderia cenocepacia BC2L-C is a super

lectin with dual specificity and proinflammatory activity. PLoS

Pathog. (2011) 7 e1002238.

30 Silva L.M., Lima V., Holanda M.L., Pinheiro P.G., Rodrigues J.

A., Lima M.E., et al. Antinociceptive and anti-inflammatory

activities of lectin from marine red alga Pterocladiella

capillacea. Biol. Pharm. Bull. (2010) 33 830–835.

31 Pajic I., Kljajic Z., Dogovic N., Sladic D., Juranic Z., Gasic M.J.

A novel lectin from the sponge Haliclona cratera: isolation,

characterization and biological activity. Comp. Biochem.

Physiol. C Toxicol. Pharmacol. (2002) 132 213–221.

32 Dresch R.R., Zanetti G.D., Lerner C.B., Mothes B., Trindade V.

M., Henriques A.T., et al. ACL-I, a lectin from the marine

sponge Axinella corrugata: isolation, characterization and

chemotactic activity. Comp. Biochem. Physiol. C Toxicol.

Pharmacol. (2008) 148 23–30.

33 Rabelo L., Monteiro N., Serquiz R., Santos P., Oliveira R.,

Oliveira A., et al. A Lactose-binding lectin from the marine

sponge Cinachy Chaetopterus variopedatus rella apion (Cal)

induces cell death in human cervical adenocarcinoma cells.

Marine Drugs (2012) 10 727–743.

34 Wang J.H., Kong J., Li W., Molchanova V., Chikalovets I.,

Belogortseva N., et al. A beta-galactose-specific lectin isolated

from the marine worm Chaetopterus variopedatus possesses

anti-HIV-1 activity. Comp. Biochem. Physiol. C Toxicol.

Pharmacol. (2006) 142 111–117.

35 Banerjee S., Chaki S., Bhowal J., Chatterjee B.P. Mucin binding

mitogenic lectin from freshwater Indian gastropod Belamyia

bengalensis: purification and molecular characterization. Arch.

Biochem. Biophys. (2004) 421 125–134.

36 Gowda N.M., Goswami U., Khan M.I. Purification and

characterization of a T-antigen specific lectin from the coelomic

fluid of a marine invertebrate, sea cucumber (Holothuria

scabra). Fish Shellfish Immunol. (2008) 24 450–458.

37 Gowda N.M., Goswami U., Khan M.I. T-antigen binding lectin

with antibacterial activity from marine invertebrate, sea

cucumber (Holothuria scabra): possible involvement in

differential recognition of bacteria. J. Invertebr. Pathol.

(2008) 99 141–145.

38 Kouriki-Nagatomo H., Hatakeyama T., Jelokhani-Niaraki M.,

Kondo M., Ehara T., Yamasaki N. Molecular mechanism for

pore-formation in lipid membranes by the hemolytic lectin

CEL-III from marine invertebrate Cucumaria echinata. Biosci.

Biotechnol. Biochem. (1999) 63 1279–1284.

39 Aragao K.S., Satre M., Imberty A., Varrot A. Structure

determination of Discoidin II from Dictyostelium discoideum

and carbohydrate binding properties of the lectin domain.

Proteins (2008) 73 43–52.

40 Brooks S.A., Wilkinson D. Validation of a simple avidin-biotin

detection method for Helix pomatia lectin (HPA) binding as a

prognostic marker in cancer. Acta Histochem. (2003) 105

205–212.

41 Mody R., Joshi S., Chaney W. Use of lectins as diagnostic and

therapeutic tools for cancer. J. Pharmacol. Toxicol. Methods

(1995) 33 1–10.

42 Debray H., Decout D., Strecker G., Spik G., Montreuil J.

Specificity of twelve lectins towards oligosaccharides and

glycopeptides related to N-glycosylproteins. Eur. J. Biochem.

(1981) 117 41–55.

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

12 R.M. Moura et al.

43 Benevides N.M.B., Holanda M.L., Melo F.R., Freitas A.L.P.,

Sampaio A.H. Purification and partial characterisation of the

lectin from the marine red alga Enantiocladia duperreyi (C.

Agardh) Falkenberg. Bot. Mar. (1998) 41 521–525.

44 BradfordM.M. A rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of

protein-dye binding. Anal. Biochem. (1976) 72 248–254.

45 de Souza G.E., Ferreira S.H. Blockade by antimacrophage

serum of the migration of PMN neutrophils into the inflamed

peritoneal cavity. Agents Actions (1985) 17 97–103.

46 Koster R., Anderson M., Debeer E.J. Acetic acid for analgesic

screening. Federation Proceedings (1959) 18 412.

47 Hunskaar S., Hole K. The formalin test in mice: dissociation

between inflammatory and non-inflammatory pain. Pain

(1987) 30 103–114.

48 Eddy N.B., Leimbach D. Synthetic analgesics. II.

Dithienylbutenyl- and dithienylbutylamines. J. Pharmacol.

Exp. Ther. (1953) 107 385–393.

49 Dunham N.W., Miya T.S. A note on a simple apparatus for

detecting neurological deficit in rats and mice. J. Am. Pharm.

Assoc. Am. Pharm. Assoc. (Baltim) (1957) 46 208–209.

50 Capaz F.R., Vanconcellos L.E., De Moraes S., Neto J.P. The open

field: a simple method to show ethanol withdrawal symptoms.

Arch. Int. Pharmacodyn. Ther. (1981) 251 228–236.

51 Bradley P.P., Priebat D.A., Christensen R.D., Rothstein G.

Measurement of cutaneous inflammation: estimation of

neutrophil content with an enzyme marker. J. Investig.

Dermatol. (1982) 78 206–209.

52 Green L.C.,Wagner D.A., Glogowski J., Skipper P.L.,Wishnok J.

S., TannenbaumS.R. Analysis of nitrate, nitrite, and [15N]

nitrate in biological fluids. Anal. Biochem. (1982)126131–138.

53 Hunskaar S., Fasmer O.B., Hole K. Formalin test in mice, a

useful technique for evaluating mild analgesics. J. Neurosci.

Methods (1985) 14 69–76.

54 Ikeda Y., Ueno A., Naraba H., Oh-ishi S. Involvement of

vanilloid receptor VR1 and prostanoids in the acid-induced

writhing responses of mice. Life Sci. (2001) 69 2911–2919.

55 Shibata M., Ohkubo T., Takahashi H., Inoki R. Modified

formalin test: characteristic biphasic pain response. Pain

(1989) 38 347–352.

56 MojicaE.R.E.,Merca F.E., PhilippinesUniv. Los Banos, College,

Laguna. Partial characterizationof lectin from the internal organs

of black sea cucumber (Holothuria atra Jaeger).Asian Inter. J. Life

Sci. (of publication Jul–Dec2006) v.15(2) p. 173–182.

57 Coelho M.B., Desouza I.A., Freire M.G., Marangoni S.,

Antunes E., Macedo M.L. Neutrophil migration in mice

induced by a mannose-binding lectin isolated from Annona

coriacea seeds. Toxicon (2006) 48 529–535.

58 Pereira-da-Silva G., Moreno A.N., Marques F., Oliver C.,

Jamur M.C., Panunto-Castelo A., et al. Neutrophil activation

induced by the lectin KM+ involves binding to CXCR2.

Biochim. Biophys. Acta (2006) 1760 86–94.

59 Collier H.O., Dinneen L.C., Johnson C.A., Schneider C. The

abdominal constriction response and its suppression by

analgesic drugs in the mouse. Br. J. Pharmacol. Chemother.

(1968) 32 295–310.

60 Tjolsen A., Berge O.G., Hunskaar S., Rosland J.H., Hole K. The

formalin test: an evaluation of the method. Pain (1992) 51 5–17.

61 Malmberg A.B., Yaksh T.L. The effect of morphine on

formalin-evoked behaviour and spinal release of excitatory

amino acids and prostaglandin E2 using microdialysis in

conscious rats. Br. J. Pharmacol. (1995) 114 1069–1075.

62 Omote K., Kawamata T., Kawamata M., Namiki A. Formalin-

induced release of excitatory amino acids in the skin of the

rat hindpaw. Brain Res. (1998) 787 161–164.

63 Santos A.R., Calixto J.B. Further evidence for the involvement

of tachykinin receptor subtypes in formalin and capsaicin

models of pain in mice. Neuropeptides (1997) 31 381–389.

64 Levine J.D., Gooding J., Donatoni P., Borden L., Goetzl E.J. The

role of the polymorphonuclear leukocyte in hyperalgesia. J.

Neurosci. (1985) 5 3025–3029.

65 Levine J.D., Lau W., Kwiat G., Goetzl E.J. Leukotriene B4

produces hyperalgesia that is dependent on

polymorphonuclear leukocytes. Science (1984) 225 743–745.

66 Bennett G., al-Rashed S., Hoult J.R., Brain S.D. Nerve growth

factor induced hyperalgesia in the rat hind paw is dependent

on circulating neutrophils. Pain (1998) 77 315–322.

67 Foster P.A., Costa S.K., Poston R., Hoult J.R., Brain S.D.

Endothelial cells play an essential role in the thermal

hyperalgesia induced by nerve growth factor. FASEB J.

(2003) 17 1703–1705.

68 Lavich T.R., Siqueira Rde A., Farias-Filho F.A., Cordeiro R.S.,

Rodrigues e Silva P.M., Martins M.A. Neutrophil infiltration is

implicated in the sustained thermal hyperalgesic response

evoked by allergen provocation in actively sensitized rats.

Pain (2006) 125 180–187.

69 Ribeiro R.A., Vale M.L., Thomazzi S.M., Paschoalato A.B.,

Poole S., Ferreira S.H., et al. Involvement of resident

macrophages and mast cells in the writhing nociceptive

response induced by zymosan and acetic acid in mice. Eur. J.

Pharmacol. (2000) 387 111–118.

70 Cunha T.M., Verri W.A. Jr, Schivo I.R., Napimoga M.H.,

Parada C.A., Poole S., et al. Crucial role of neutrophils in the

development of mechanical inflammatory hypernociception. J.

Leukoc. Biol. (2008) 83 824–832.

71 Nunes B.S., Rensonnet N.S., Dal-Secco D., Vieira S.M., Cavada

B.S., Teixeira E.H., et al. Lectin extracted from Canavalia

grandiflora seeds presents potential anti-inflammatory and

analgesic effects. Naunyn Schmiedebergs Arch Pharmacol.

(2009) 379 609–616.

72 Bitencourt Fda S., Figueiredo J.G., Mota M.R., Bezerra C.C.,

Silvestre P.P., Vale M.R., et al. Antinociceptive and anti-

inflammatory effects of a mucin-binding agglutinin isolated

from the red marine alga Hypnea cervicornis. Naunyn

Schmiedebergs Arch Pharmacol. (2008) 377 139–148.

73 Nemirovsky A., Chen L., Zelman V., Jurna I. The

antinociceptive effect of the combination of spinal morphine

with systemic morphine or buprenorphine. Anesth. Analg.

(2001) 93 197–203.

74 Soja P.J., Taepavarapruk N., Pang W., Cairns B.E., McErlane

S.A., Fragoso M.C. Transmission through the dorsal

spinocerebellar and spinoreticular tracts: wakefulness versus

ª 2012 The Authors Fundamental and Clinical Pharmacology ª 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology

HGA the first invertebrate lectin with anti-inflammatory effects 13