gy, Part A 145 (2006) 517–523www.elsevier.com/locate/cbpa

Comparative Biochemistry and Physiolo

CvL, a lectin from the marine sponge Cliona varians: Isolation,characterization and its effects on pathogenic

bacteria and Leishmania promastigotes

Raniere M. Moura a, Alexandre F.S. Queiroz a, Jacy M.S.L.L. Fook b, Anny S.F. Dias c,Norberto K.V. Monteiro c, Jannisson K.C. Ribeiro c, Gioconda E.D.D. Moura c,

Leonardo L.P. Macedo c, Elizeu A. Santos c, Maurício P. Sales c,⁎

a Departamento de Biofísica e Farmacologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazilb Departamento de Morfologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil

c Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, 59072-970, Natal, RN, Brazil

Received 16 May 2006; received in revised form 16 August 2006; accepted 21 August 2006Available online 30 August 2006

Abstract

CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinitychromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was stronglyinhibited by monosaccharide D-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphidebridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+ dependent, stable upto 60 °C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis andStaphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmaniachagasi promastigotes were agglutinated by CvL up to 28 titer. These findings are indicative of the physiological defense roles of CvL and itspossible use in the antibiosis of bacteria and protozoa pathogenic.© 2006 Elsevier Inc. All rights reserved.

Keywords: Sponge; Cliona varians; Pathogenic bacteria; Leishmania; Invertebrate marine

1. Introduction

Marine invertebrates survive in environments rich in bacterialpopulations that may be as high as 106 and 109/mL (Austin,1988), many of which may be pathogenic. The immune defenceof these animals is non-specific with responses against microbialorganisms based on both cellular (phagocytosis, encapsulation,respiratory burst, etc.) and humoral (agglutinins, lysozomalenzymes, antimicrobial factors, etc.) activities (Chu, 1988;Canesi et al., 2002). In marine animals, humoral lectins havebeen shown to participate actively, much the same as opsonins,in the phagocytosis of non-self-cells and molecules (Bayne,1990). Lectins are ubiquitous proteins in the hemolymph and

⁎ Corresponding author. Tel.: +55 84 32153416; fax: +55 84 32119208.E-mail address: msales@cb.ufrn.br (M.P. Sales).

1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2006.08.028

cells of many invertebrates (Renwrantz, 1986; Cooper et al.,1992) with the activity based in the carbohydrate-bindingprotein that produces the agglutination of many cells, such aserythrocytes, bacteria, and others through interaction withspecific complementary ligands (Goldstein et al., 1980; Sharon,1984; Nesser et al., 1986). Moreover, there is evidence sup-porting the fact that marine invertebrate lectins are also involvedin other endogenous biological events such as biomineralization(Goldstein et al., 1980) and embryonic development (Nesseret al., 1986; Bayne, 1990). Interestingly, it has been theorizedthat some marine invertebrate lectins mediate the interactionbetween symbiont and host (Vasta, 1992). Despite their ubiquity,their functions in nature are not fully clear. They represent aheterogeneous group of oligomeric proteins that vary widely insize, structure, molecular organization, and constitution of theircombining sites. Many lectins belong to distinct protein groups

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with similar sequences and structural features. They wereinitially classified into two different groups, C- and S-typelectins, based particularly on the carbohydrate-recognitiondomain (CRD), along with overall domain organization andphysical–chemical properties, such as divalent cation depen-dence and free thiol requirement (Drickamer, 1988). Calciumdependence was the main property associated with C-typelectins, whereas thiol dependence was the characteristicassociated to the S-type group. Recently, as a consequence ofthree-dimensional structure studies and the availability ofadditional primary sequence data, a more complete classificationhas emerged. Therefore, C-type lectins have been further cha-racterized into distinct subgroups, and S-type lectins reclassifiedas galectins; but the existence of new categories was alsoestablished (Barondes et al., 1994; Drickamer, 1995; Vasta et al.,1997; Vasta et al., 1999).

In most of the publications concerning anti-pathogenicactivity in invertebrates, either single body compartments alone,such as haemolymph and egg masses, or extracts of wholebodies have been tested for antibiosis activity. Haug et al.(2004) recently showed that marine invertebrates possessantipathogen factors in several different tissues. The aim ofthe present paper was to describe the purification and cha-racterization of a galactose-binding lectin from the body of thesponge Cliona varians with respect to its chemical analysis,sugar specificities, and anti-pathogenic activities, revealing aplausible biological role of the lectin and use as an antibiotic.

2. Material and methods

2.1. Material

Papain, bovine trypsin and neuraminidase were purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). Humanerythrocyte types A, B and O was donated by the Blood Bank,Hemocentro, Natal, Brazil. Bacteria were obtained from thecollection donated by Dr. Patricia Soto-Mayor Sommer ofDepartamento de Biologia Celular e Genética of the Universi-dade Federal do Rio Grande do Norte, Natal, Brazil. Leishmaniachagasi promastigotes were donated by Dr. Paulo Andrade ofDepartamento de Genética of the Universidade Federal dePernambuco, Recife, Brazil.

2.2. Animal collection

Adult specimens of the marine sponge C. varians werecollected in the littoral of Buzios beach, Parnamirim, Brazil.Specimens were collected and transported in ice to the laboratoryand stored at �20 °C until use.

2.3. Haemagglutinating activity

The haemagglutinating activity was assayed in microtiter Vplates (Nunc Brand products, Denmark) according to a twofoldserial diluting procedure (Debray et al., 1981). The erythrocytesused were native or treated with papain and/or trypsin accordingto Benevides et al. (1998). One haemagglutinating unit (HU)

was defined as the amount of lectin able to agglutinate and henceprecipitate the erythrocytes in suspension after 30 min. To eachwell was added 25 μL of the twofold serially diluted lectinsolutions and 25 μL of treated and untreated human erythrocytes(2% v/v suspension) and allowed to incubate for 30 min at roomtemperature. The degree of haemagglutinating activity wasexamined. The controls were set up with saline and erythrocytesand 1 mg/mL of Con A solution and erythrocytes.

2.4. Purification of marine sponge C. varians lectin (CvL)

Specimens were cut into small pieces using sharp scissors.The pieces were ground 5–6 times with a mixer machine andstirred vigorously and extracted (1:2, w/v) with 0.05 M Tris–HCl buffer pH 7.5, for 2 h at room temperature. Aftercentrifugation for 30 min at 12,000×g at 4 °C, the supernatant(crude extract) was precipitated with acetone at 0.5 vol, 1.0 voland 2.0 vol. These fractions (F1, F2 and F3) were then freezer-dried and submitted to assays. The F2 fraction showed a higherlevel of haemagglutinating activity to human papainizederythrocytes type A. This fraction was applied to a SepharoseCL 4B (5.5×3.5 cm) affinity chromatography, equilibrated with50 mM Tris–HCl, 20 mM CaCl2 buffer, pH 8.0. The retainedproteins were eluted with 50 mMTris–HCl, 100 mMEDTA, pH8.0, at a flow rate of 30 mL h−1. The retained peak, denominatedCvL, was pooled and subjected to further analysis. Proteincontent of all fractions was measured as described by Bradford(1976) and chromatography was monitored at 280 nm.

2.5. Carbohydrate specificity

The sugar specificity of the lectin was determined in the plateassay as described above. All inhibitors to be tested weredissolved in 150 mMNaCl at an initial concentration of 200 mMfor monosaccharides (D-galactose, D-glucose, D-fructose, D-fucose, D-ribose, D-arabinose, D-xilose, D-N-acetyl-glucosamineand D-glucuronic acid) and disaccharides (maltose, sucrose andlactose). An equal volume of the lectin solution was added to25 μL of the twofold serially diluted inhibitor solutions and theplate was incubated for 1 h at room temperature. Twenty-fivemicroliters of human erythrocytes (2% v/v suspension) wasadded to each well and allowed to incubate for 30 min at roomtemperature. The degree of haemagglutination was examinedand the maximum dilution of the inhibitor solution showinginhibition was recorded. The controls were set up with saline anderythrocytes, sugar and erythrocytes, and lectin and erythro-cytes. Results were expressed as the minimal sugar orglycoprotein concentration required to inhibit haemagglutinat-ing doses of the lectin.

2.6. Effects of metal ions, temperature and pH onhaemagglutinating activity

The effect of pH on the lectin activity was studied byhaemagglutinating assay of the lectin after dialysis againstbuffers of different pH ranging from pH 2.5 to pH 10.5containing 100 mM CaCl2 for 3 h. 50 mM glycine–HCl buffer

Table 1Purification process of CvL by affinity chromatography on Sepharose 4B

Fraction Total protein(mg)

TotalHU

Specific activity(HU/mg)

Purification(x)

Recovery(%)

Crude extract 135 128 0.95 1.0 100F2.0 19 256 13.5 14.2 14CvL 1.4 256 182.9 192.5 1

One haemagglutinating unit (HU) was defined as the amount of lectin able toagglutinate and hence precipitate the erythrocytes in suspension after 30 min.

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(pH 2.5), 50 mM. Acetate buffer (pH 4.5), 50 mM phosphatebuffer (pH 6.5), 50 mM Tris–HCl buffer (pH 8.5), and 50 mMglycine–NaOH buffer (pH 10.5) were used.

The effect of divalent metal ions on the haemagglutinatingactivity of the lectin was assessed as follows. The lectin wasdialyzed exhaustively against 50 mM EDTA followed bydialysis against 50 mM Tris–HCl buffer, pH 8.0. The haema-gglutinating activity was tested in the presence and in theabsence of 100 mM Ca2+, Mg2+ and Mn2+.

To study the effects of temperature on haemagglutinatingactivity, lectin (0.15 mg/mL) was serially diluted, incubated at−10, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 °C for 15, 30and 60 min at each temperature and assayed after thehaemagglutinating activity.

Fig. 1. (A) Elution profile on Sepharose CL 4B of CvL. Approximately 1.6 mgof F1.0 was applied in the column (5.5×3.5 cm) equilibrated with 50 mM Tris–HCl buffer, containing 20 mM CaCl2, pH 7.5. The lectin was eluted with 50 mMTris–HCl buffer, containing 20 mM EDTA pH 7.5 and the fractions (1.5 mLeach) and fractions were monitored at 280 nm. The haemagglutinating activitywas detected in the presence of 2% human erythrocytes type A. (B) SDS-PAGEanalysis of the (M) Molecular Weight Markers (kDa): β-galactosidase (116.0),BSA (66.2), ovalbumin (45.0), Lactate dehydrogenase (35.0), Restrictionenzyme Bsp98I (25.0), β-lactoglobulin (18.4) and Lysozyme (14.4); CvL;βCvL in presence of β-mercaptoethanol (0.1 M). Proteins were stained withCoomassie blue and after with silver.

2.7. Sub-units determination, molecular mass estimation andstaining glycoproteins

SDS polyacrylamide (7.5% and 12%) gel electrophoresis(SDS-PAGE) in the absence and presence of β-mercaptoethanol(0.1 M) was conducted as described by Laemmli (1970) toestimate the molecular mass of lectin and its sub-units bycomparing of mobility of bands with protein molecular weightmarkers (kDa): β-galactosidase (116.0), BSA (66.2), ovalbumin(45.0), Lactate dehydrogenase (35.0), Restriction enzymeBsp98I (25.0), β-lactoglobulin (18.4) and Lysozyme (14.4).Proteins were detected by staining with 0.1% Coomassiebrilliant blue R-250 followed by silver staining according toBlum et al. (1987), with modifications. Also, the molecular massof CvL was estimated by Sephacryl S-200 gel filtration column(75×1.4 cm) calibrated with protein markers (kDa): potato β-amylase (Mr 150), bovine serum albumin (Mr 67), Hemoglobin(Mr 55) and trypsin inhibitor (Mr 21.5).

Staining glycoproteins in SDS-Polyacrylamide Gels wasdescribed by Zacharius et al. (1969).

2.8. Antibacterial activity test

Four strains of pathogenic bacteria (Escherichia coli, Bacillussubtilis, Pseudomonas aeruginosa and Staphylococcus aureus)were selected for CvL agglutination. Bacteria were grown in LBbroth (10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl, 1 LH2O, pH 7·0). Cells in the exponential phase (15 h)were collectedand washed three times with 0.15 M NaCl solution bycentrifugation at 6000×g for 10 min. The antibacterial activitieswere determined by continuous monitoring of bacterial growth.The test was performed in 1 mL plastic cuvette (Labcraft), inwhich 25 μg mL−1, 50 μg mL−1 and 100 μg mL−1 of CvL wereincubated with 100 μL of a suspension of bacteria culture dilutedto a starting concentration of 5×103 cells per cuvette. The growthchamber was maintained at 37 °C during the incubation period.The absorbance was measured at 1 h intervals for 4 h by aturbidometric method. Controls were added to assays: (1) bacteriaplus 200 μL of 150 mM NaCl solution; (2) bacteria plus 25 μgmL−1, 50 μg mL−1 and 100 μg mL−1 of BSA; (3) bacteria plus25μgmL−1, 50μgmL−1 and 100μgmL−1 of CvL pre-incubatedin 200 mM galactose solution.

2.9. L. chagasi promastigotes agglutination test

L. chagasi prosmatigotes were briefly maintained inmodified Dwyer medium (Dwyer, 1972), with beef extract

Fig. 2. Determination of molecular weight of CvL by gel filtration on SephacrylS-300 chromatography. Proteins were applied in the column (127×2.6 cm)equilibrated with 50 mM Tris–HCl buffer, containing 20 mM CaCl2, pH 7.5.The eluted protein fractions (1.5 mL each) were monitored at 280 nm. Proteinmolecular weight standards were: (a) potato β-amylase (Mr 150), (b) bovineserum albumin (Mr 67), (c) Hemoglobin (Mr 55) and (d) trypsin inhibitor (Mr21.5).

Fig. 3. General properties of the CvL. The effects of (A) temperature, (B) pH and(C) Ca2+ on the haemagglutination activity of CvL.

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substitution. Defibrinated rabbit blood, yeast extract (5.0 g/l),and tryptone (0.1 g/l) were added to the basic medium. Massproduction of the parasite was obtained by inoculating 1 l ofRPMI 1640 to HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and 20 mL of rich promastigote mainte-nance culture were added to the fetal bovine serum (FBS). Afterharvest, the promastigotes were washed several times with coldLocke solution, treated at 37 °C with 0.4% trypsin for 45 min,and fixed for 20 h at 4 °C with 2% (w/v) formaldehyde in Lockesolution. After being washed in saline, the parasites werestained with 0.1% Coomassie brilliant blue for 90 min. Theparasite suspension was filtered through nylon gauze, and theconcentration was adjusted to 5×107 cells/mL in 0.4% (w/v)formaldehyde. The agglutinating activity was assayed inmicrotiter V plates (Nunc Brand products, Denmark) accordingto a twofold serial diluting procedure. To each well were added25 μL (1 mg mL−1) of the twofold serially diluted lectinsolutions and 25 μL of blue promastigotes and allowed toincubate for 24 h at 5 °C. The degree of agglutination wasexamined. Two controls were added: (1) saline and bluepromastigotes; (2) CvL plus 200 mM D-galactose solution andblue promastigotes.

3. Results

3.1. Lectin isolation and CvL purification

Crude soluble protein extract obtained from marine spongewas initially precipitated at 0.5 vol, 1.0 vol and 2.0 vol withacetone and three protein fractions (F1, F2 and F3) wereobtained. The F2 protein fraction obtained showed stronghaemagglutining activity to papainized type A erythrocytesfollowed by papainized type B erythrocytes, while the otherfractions exhibited low activity. The haemagglutining activity

of F2 was activated by ions Ca2+. The F2 was then applied to aSepharose affinity column and the retained peak was submittedto haemagglutining test (Fig. 1A). This purification procedurefor marine sponge C. varians was observed by SDS-PAGE(Fig. 1B) and resulted in a high purification of 192.5 fold with a1% yield (Table 1).

3.2. Physical and chemical analysis of the CvL

Electrophoretic analysis of CvL in the absence of a reducingagent (β-mercaptoethanol), showed one protein band withmolecular mass of approximately 106 kDa, which aftertreatment with β-mercaptoethanol decomposed to four subunitsof 28 kDa (Fig. 1B). The glycoprotein staining procedureshowed that CvL was marked as a magenta banding, a patterncharacteristic of a glycoprotein (data not showed).

Table 2Inhibitory effect of monosaccharides, disaccharides and glycoprotein onhaemagglutinating activity of CvL

Inhibitor compound MIC (mM)

MonosaccharidesD-Galactose 6.25D-Glucose 25D-Fructose NIL-Fucose NID-Ribose NID-Glucuronic acid 200D-Arabinose NID-Xilose NID-N-acetyl-glucosamine NI

DisaccharidesMaltose 25Sucrose 12.5Lactose 12.5

MIC, minimum inhibitory concentration; NI, sugar not inhibitory until aconcentration of 200 mM.

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The relative molecular mass of the native lectin wasestimated at 114 kDa by gel filtration on a calibrated SephacrylS-200 column (Fig. 2).

The study of the temperature effect on CvL showed that thelectin was stable at 60 °C (Fig. 3A). Preincubation of the

Fig. 4. Growth curves of B. subtilis (A) and S aureus (B) in the presence of theCliona varians lectin (CvL). (○) control, bacteria plus 200 μL 0.15 M NaClsolution; (▲) control bacteria plus with BSA (100 μg/mL); (♦) bacteria plusCvL (25 μg/mL), (▪) CvL (50 μg/mL) and (•) CvL (100 μg/mL). Bacterialgrowth was measured from absorbance at 600 nm. Each point represents themean of 3 estimates.

Fig. 5. Agglutination test for Leishmania chagasi promastigotes stained with0.1% Coomassie brilliant blue. (A) Control, promastigotes plus 25 μl 0.15 MNaCl solution; (B) promastigotes plus 25 μg CvL and 25 μL 0.2 M D-galactosesolution; (C) promastigotes plus 25 µg CvL. Magnification: 40×. bar: 250 μm.

lectin in 6.0–8.0 pH range did not affect haemagglutinatingactivity (Fig. 3B).

Haemagglutinating activity of CvL was found to be Ca2+

dependent and an inhibition by 50 mM EDTA solution wasreverted at 20 mM of CaCl2 prepared in 150 mM NaCl solution(Fig. 3C).

3.3. Lectin specificity

Haemagglutinating activity of the CvL towards papainizedType A erythrocytes was mostly inhibited by D-galactose(Table 2).

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3.4. Antibacterial and anti-Leishmania effects

The CvL Antibacterial activity was observed against grampositive bacteria, such as B. subtilis and S. aureus. The resultsshowed that CvL, at a concentration of 25 μg/mL, exhibitshigh antibacterial activity (75%) on B. subtilis, which increaseswith higher concentrations; reaching 90% of inhibition with100 μg/mL, at 4 h (Fig. 4A). High antibacterial activity was alsoobserved when CvL was incubated with S. aureus, reducingbacteria growth by 90% at 50 μg/mL, at 4 h (Fig. 4B). The inhi-bitory effect of the lectin on the gram negative bacteriaE. coli andP. aeruginosa was comparatively low. The agglutination test forL. chagasi promastigotes stained with 0.1% Coomassie brilliantblue showed that CvL was capable, according to a twofold serialdiluting procedure, agglutinated up to 8 AU (agglutinating unit)titer, corresponding to the agglutination of 106 cells by 1 μg ofCvL. The binding of CvL and promastigotes was not observedwhen the mixture was incubated in presence of D-galactose(Fig. 5A, B and C).

4. Discussion

Bioactive carbohydrates, proteins, peptides, and secondarymetabolites have been isolated from various marine invertebrateclasses, including the bioprospection of Porifera, which may beof tremendous potential benefit for humans. Among thesebioactive molecules, lectins are proteins involved in the non-immune responses in invertebrate marines and their effects onthe antibiosis of pathogenic organisms have been reported. Inthis study, a lectin with molecular mass around 114 kDa, formedby four subunits of 28 kDa linked by disulphide bridges, wasisolated and purified to homogeneity from the sponge marine C.varian. The lectin, which was denominated CvL, exhibited Aerythrocyte papainized specificity. CvL is a glycoprotein stableat pH range 6 to 8 and up to a temperature of 50 °C. The activityof this lectin was also found to be Ca2+ dependent, as evidencedby complete activity loss in the presence of EDTA and theaddition of Ca2+ ions regains the activity of the CvL. Thepresence of glycosylation and disulphide bridges in the proteinwas also indicative that CvL should be included in the C typelectin family, such as Cinachyrella alloclada (Atta et al., 1989),Pellina semitubulosa (Engel et al., 1992), Axinella polypoide(Buck et al., 1992) and Haliclona cratera (Pajic et al., 2002).

The binding property of the CvL was found to be preferentialfor galactose. A considerable number of lectins, including thoseisolated from sponges, were reported to react with D-galactose(Bretting et al., 1981; Schröder et al., 1990). Lectins with affinityfor galactose appear to have important roles in modulatingimmune responses in marine animals (Yousif et al., 1994; Mistryet al., 2001; Kurata and Hatai, 2002). It has also been reportedthat several lectins isolated from various marine invertebratescan inhibit the growth of various bacterial pathogens and fungi(Tateno et al., 2002; Tasumi et al., 2004). CvL exhibited a stronganti-bacterial effect on pathogenic gram positive bacteria, suchas B. subtilis and S. aureus whereas no effects were observed onpathogenic gram negative bacteria. This pattern of differentia-tion was observed among lectins from diverse origins because

almost all microorganisms express surface-exposed carbohy-drates as a potential lectin-reactive specific site. This property isexemplified in the invertebrate marine Tachypleus tridentatus,where several lectins, denominated tachylectins 1, 2, 3 and 4,were purified from their hemolymph. Tachylectin 1 was able toinhibit the growth of gram-negative bacteria (Saito et al., 1995).Tachylectin 2 had a binding specificity for N-acetylglucosamine(GlcNAc) and N-acetylgalactosamine (GalNAc) and promotedthe agglutination of certain strains of gram-positive Staphylo-coccus (Okino et al., 1995; Kawabata and Iwanaga, 1999);Tachylectin 3 and Tachylectin 4 recognized the LPS of severalgram-negative bacteria (Saito et al., 1997; Inamori et al., 1999).The binding of CvL to galactose sites in bacterial surface couldexplain deterrent effects found in agglutinating tests with gram-positive bacteria. The property of these lectins to select andcomplex with microbial glycoconjugates has made it possible toemploy the proteins as probes and sorbents for whole cells,mutants and numerous cellular constituents and metabolites.

The few past years have witnessed an increasing interest inlectin–parasite interactions. This is because lectins havebecome valuable tools for studying the insertion, fate,distribution and function of glycoconjugates on and in parasites;for example, lectins are useful in defining various developmen-tal parasite cycles. This experiment showed that CvL hadagglutinating activity on promastigotes of L. chagasi. Thiseffect revealed that galactose receptors for lectin binding arepresent in this parasite stage. Parasite lectin specificity has beenextensively studied in Trypanosomatids with respect to theirlectin receptors (Jacobson, 1994; Gazzinelli et al., 1991;Schottelius and Alsien, 1994), for example, lectin 1B 4 ofGriffonia simplicifolia, has been utilized to define terminal α-galactosyl-bearing glycoconjugates on T. cruzi, Leishmaniabraziliensis and Leishmania mexicana parasites (Petri et al.,1989; Souto-Padron et al., 1994). This is important because of thepresence of anti-galactose antibodies that are found in increasedamounts in the sera of patients infected with these parasites.

These findings showed that the isolation and characterizationof proteins from marine animals has turned into a fast growingfield in the life sciences because many marine bioactivemolecules are attracting more and more attention due to theirextensive physiological, biological and pharmacological use.The importance of marine biotechnology has grown immenselybecause of the achievements of these natural marine products.

Acknowledgments

This work was supported by Brazilian Agencies: CAPESand CNPq. The authors thank Dr. Rosana Lucena de Sá Leitão,Dr. Dulce H. Seabra de Souza Silva and Silene Telma Lima deSantana, pharmacists from Hemonorte, Natal, RN, Brazil, forthe generous blood bag donation.

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