Artigos científicos sobre lectinas, Pesquisas de Bioquímica. Universidade Federal do Ceará (UFCE)
r_mulo_farias19 de Outubro de 2017

Artigos científicos sobre lectinas, Pesquisas de Bioquímica. Universidade Federal do Ceará (UFCE)

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A chromophore-containing agglutinin from Haliclona manglaris: Purification and biochemical characterization


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International Journal of Biological Macromolecules 72 (2015) 1368–1375

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

chromophore-containing agglutinin from Haliclona manglaris: urification and biochemical characterization

ômulo Farias Carneiroa, Alexandra Sampaio de Almeidaa, Arthur Alves de Meloa, aniel Barroso de Alencara, Oscarina Viana de Sousab, Plínio Delatorrec, yria Santiago do Nascimentod, Silvana Saker-Sampaioa, Benildo Sousa Cavadad, elso Shiniti Naganoa, Alexandre Holanda Sampaioa,∗

Laboratório de Biotecnologia Marinha–BioMar-Lab, Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Campus do Pici s/n, bloco 871, 0440-970 Fortaleza, CE, Brazil Instituto de Ciências do Mar–Labomar, Universidade Federal do Ceará, Av. da Aboliç ão, 3207, 60165-081 Fortaleza, CE, Brazil Departamento de Biologia Molecular, Universidade Federal da Paraíba, João Pessoa, PB, Brazil Laboratório de Moléculas Biologicamente Ativas—BioMol-Lab, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus o Pici, s/n bloco 907, 60440-970 Fortaleza, CE, Brazil

r t i c l e i n f o

rticle history: eceived 8 September 2014 eceived in revised form 2 October 2014 ccepted 3 October 2014 vailable online 13 October 2014


a b s t r a c t

A new chromophore-containing agglutinin (Haliclona manglaris agglutinin (HMA)) was isolated from the tropical sponge H. manglaris. HMA was purified by a combination of hydrophobic interaction chro- matography and ion exchange chromatography. Native HMA is a heterotrimer formed by two -chains (15 kDa) and one -chain (22 kDa). HMA is a glycoprotein and possesses three intrachain disulfide bonds. Hemagglutinating activity of HMA was stable at neutral pH and temperatures up to 60 ◦C. HMA was only inhibited by thyroglobulin. Mass spectrometry sequencing and Edman degradation revealed a unique

ectin hromophore ponge

amino acid sequence of about 30%. Moreover, HMA has an organic chromophore of 581 Da, and this char- acteristic seems to be important to its antioxidant activity. Interestingly, while HMA showed no toxicity against Artemia nauplii and was unable to agglutinate bacterial cells, it did show a high capacity to pro- tect -carotene against oxidation. Thus, our findings suggest the putative involvement of HMA in the protection of the sponge against oxidation.

© 2014 Elsevier B.V. All rights reserved.


Lectins are carbohydrate-binding proteins that are not involved n carbohydrate metabolism, and do not belong to any of the main lasses of immunoglobulins [1]. Lectins play several physiologi- al roles, and they are involved in diverse biological processes in nimals, such as symbiosis, infection defense, apoptosis and sugar raffic [2–4].

Recently, many lectins have been isolated from marine sources 5–7]. In particular, marine invertebrates have attracted some nterest. In these organisms, lectins play a crucial role in innate mmune response. They act as opsonins, recognizing foreign sub-

tances through binding to their carbohydrate components and riggering phagocytosis of pathogens by scavenger cells [8].

∗ Corresponding author. Tel.: +55 85 33669728; fax: +55 85 33669728. E-mail address: (A.H. Sampaio).

ttp:// 141-8130/© 2014 Elsevier B.V. All rights reserved.

The physiological role of a lectin is typically mediated by the car- bohydrate recognition domain (CRD) and by their ability to bind to glycosylated epitopes or other carbohydrate structures. Neverthe- less, only a few studies have reported on the ability of lectins to bind to non-glycosylated molecules [9,10]. For instance, the third lectin isolated from the marine sponge Haliclona caerulea (H-3) is a blue lectin that recognizes N-acetyl-galactosamine. H-3 has color because it interacts with a hydrophobic chromophore of 597 Da (chromophore-597) via a domain thus far uncharacterized [11].

The amino acid sequence of H-3 showed high identity with a putative protein encoded in the genome of the sponge Amphimedon queenslandica, pAqP, but it showed no significant similarity with any other lectins [11]. Thus, H-3 could not be grouped in any family of animal lectins, e.g., C-type, F-type, L-type, RBL or Galectins.

Porifera represents a basal group of metazoans, and lectins iso-

lated from sponges are among the most ancient protein known. Only a few structural studies have reported on sponge lectins. Nev- ertheless, some sponge lectins seem to play a physiological role in cellular re-aggregation [12], symbiosis [13] and growth regulation


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14]. However, up to now, no chromophore-containing lectin has een reported in sponge or other animals. Consequently, the physi- logical role of chromophore-containing lectins remains unknown. Therefore, we have searched for lectins similar to H-3, i.e., those

aving significant amino acid sequence identity and those interac- ing with chromophores. Here we report a new agglutinin isolated rom the tropical sponge Haliclona manglaris (HMA). This protein hared properties similar to those of H-3, including a chromophore, wo polypeptide chains and glycosylation, but, curiously, HMA and -3 do not share sequence similarity. We also looked for evidence upporting the physiological roles of these proteins.


.1. Animal collection

Specimens of the marine sponge H. manglaris were collected in he intertidal zone of Icaraí Beach, Amontada, Ceará State, Brazil. resh sponges were transported on ice to the laboratory and stored t −20 ◦C until use. The species was identified, and a voucher as deposited (identification number: MNRJ 15936) at the Museu acional-UFRJ, Rio de Janeiro, Brazil.

.2. Isolation of H. manglaris agglutinin

Frozen sponges were cut into small pieces, triturated into pow- er, and extracted (1:2, w/v) with 20 mM acetate buffer, pH 5.5 (AB). he mixture was strained through nylon tissue and clarified by cen- rifugation for 20 min at 10,000 × g at 4 ◦C. The supernatant (crude xtract) was collected and assayed for hemagglutinating activity see below) and protein concentration [15].

Ammonium sulfate was added to the crude extract to achieve he final concentration of 500 mM, and the suspension was left or 4 h at room temperature. The precipitated proteins were emoved by centrifugation for 20 min at 10,000 × g at 4 ◦C, and he supernatant was loaded on a Phenyl-Sepharose 6B column 1.0 cm × 5.0 cm) equilibrated with 500 mM of ammonium sulfate n AB. The column was washed with the same buffer at a flow rate f 2 mL min−1 until the column effluents showed absorbance of less han 0.02 at 280 nm. Two adsorbed fractions (P1 and P2) were eluted ith 200 mM of ammonium sulfate in AB and ethanol 20%, respec-

ively. The chromatography was monitored at 280 nm, and 3-mL ractions were manually collected and tested for hemagglutinating ctivity. The active fraction (P2) was dialyzed against deionized water,

reeze-dried, solubilized in a small volume of 20 mM Tris–HCl uffer, pH 7.6 (TB), and loaded onto a DEAE-Sephacel column 1.0 cm × 3.0 cm) previously equilibrated with TB. The flow rate as adjusted to 1 mL min−1, and the column was washed with TB

ollowed by elution of two adsorbed fractions (D1 and D2) with 00 mM and 1 M of NaCl in TB, respectively. The chromatography as monitored at 280 nm and 660 nm, and 1-mL fractions were ollected. Fraction D1 showed hemagglutinating activity and was ermed H. manglaris agglutinin (HMA).

.3. Purification of H-3, the blue lectin from Haliclona caerulea

H. caerulea was collected at Pacheco Beach, Caucaia, Ceará State, razil. Purified H-3 was obtained using the method previously escribed by Carneiro et al. [11].

.4. Hemagglutinating activity and Hemagglutination inhibitory


The hemagglutination tests were performed on microtiter plates ith V-bottom wells using the two-fold serial dilution method. One

ical Macromolecules 72 (2015) 1368–1375 1369

hemagglutinating unit (HU) was defined as the amount of lectin able to agglutinate and, hence, precipitate erythrocytes in a sus- pension after 1 h. Human (A, B and O type) and rabbit erythrocytes were used in native form and treated with papain and trypsin.

A hemagglutination inhibition assay was performed using the standard procedure [16]. The following carbohydrates and glycoproteins were used: d-fructose, d-fucose, d- galactose, d-glucose, d-mannose, methyl--d-galactopyranoside, methyl--d-glucopyranoside, N-Acetyl-d-galactosamine, N- acetyl-d-glucosamine, N-Acetyl-d-mannosamine, d-Sucrose, -lactose, -lactose, lactulose, tyroglobulin, ovomucoid and porcine stomach mucin (PSM).

2.5. Molecular mass estimation, sugar content and absorbance spectrum

The molecular mass of HMA under denaturing condition was estimated by SDS-PAGE in 15% gel in the presence and absence of -mercaptoethanol, followed by staining with Coomassie Brilliant Blue, as described by Laemmli [17]. Homogeneity of the native HMA was evaluated by N-PAGE 12%.

The apparent molecular mass of the native HMA was esti- mated by size exclusion chromatography on BiosuiteTM 250 HR SEC (0.78 × 30 cm, 5 m particle size) column coupled to an Acquity UPLCTM system (Waters Corp.). The column was equilibrated with 50 mM Tris–HCl, pH 7.2, containing NaCl 500 mM, and calibrated with conalbumin (75 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa).

Neutral carbohydrate content in HMA was evaluated as described by Dubois et al. [18], using lactose as the standard. Glyco- proteins in SDS-PAGE were stained with periodic acid-Schiff (PAS), as described by Zacharius et al. [19].

A wavescan was performed using the UltrospecTM 2100Pro UV–Vis Spectrophotometer with wavelength ranging from 190 nm to 900 nm to determine maximum absorbance of HMA.

2.6. Effects of pH, temperature and divalent cations on the hemagglutinating activity of HMA

The effects of pH, temperature, EDTA and divalent cations on lectin activity were evaluated as described by Sampaio et al. [16].

2.7. Molecular mass determination

Purified HMA was solubilized with 5% acetonitrile (ACN) con- taining 0.1% trifluoroacetic acid (TFA) and submitted to reverse phase chromatography (RPC) coupled to the Acquity system (Waters Corp., Milford, MA, USA). Sephasil Peptide C-8 10/250 col- umn (GE-Healthcare) was equilibrated and washed with ACN 5% containing TFA 0.1% at a flow rate of 1 mL min−1. Retained pro- teins were recovered by elution through a gradient of 5% to 70% of ACN containing TFA 0.1%. Absorbance of the column eluates was monitored at 216 and 280, and fractions of 0.5 mL were collected.

After separation, HMA chains were vacuum-dried and submit- ted to mass spectrometry analysis. The average molecular masses of HMA chains were determined using a hybrid Synapt HDMS mass spectrometer (Waters Corp., Milford, MA, USA). HMA chains were solubilized in 50% ACN containing 0.1% formic acid (FA). After centrifugation, protein solutions (10 pmol L−1) were separately infused at a flow rate of 1 L min−1 into a nanoelectrospray source coupled to a mass spectrometer. The instrument was calibrated with [Glu1]fibrinopeptideB fragments. Mass spectra were acquired

by scanning at m/z ranging from 500 to 4000 at 2 scan/s. The mass spectrometer was operated in positive mode, using a source tem- perature of 363 K and capillary voltage at 3.2 kV. Data collection and processing were controlled by Mass Lynx 4.1 software (Waters).

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tilled water, 2 mM ferrous chloride (FeCl2), 5 Mm ferrozine and HMA and H-3 at different concentrations. Distilled water was used for both blank sample and control, instead of ferrozine and HMA and H-3, respectively. The percentage of ferrous ion chelating activ- ity was calculated using the expression below:

370 R.F. Carneiro et al. / International Journal of

econvolution of ESI mass spectra was performed using the MaxEnt algorithm in the Mass Lynx software.

.8. Quantification of sulfhydryl groups

Free cysteine residues and total cysteine residues in HMA were uantified as described by Carneiro et al. [11].

.9. N-terminal analysis

Automated Edman degradations were performed with protein equence Shimadzu model PPSQ-31A (Shimadzu Corp., Japan). TH-amino acids from the N-terminus sequence were separated n a 2.0 × 250 mm ODS column (Wakosil) connected to a model LC- 0AT pump. The absorbance was detected at 269 nm with a UV–Vis PD-20A detector.

.10. Amino acid sequencing by tandem mass spectrometry MS/MS)

SDS-PAGE was performed as described above. After stain- ng, HMA spots ( and  chains) were excised, reduced with ithiothreithol (DTT), and carboxyamidomethylated with iodoac- tamide (IAA), as described by Shevchenko et al. [20]. Treated spots ere subjected to digestion with trypsin (Promega). Digestions ere performed in ammonium bicarbonate 50 mM at 1:50 w/w

enzyme/substrate) and maintained at 37 ◦C for 16 h. The digestions were stopped with 2 L of 2% FA. The samples

ere washed four times with 5% FA in 50% ACN. The supernatants ere collected and transferred to fresh tubes, pooled, vacuum- ried, solubilized in 20 L of 0.1% FA, and centrifuged at 10000 × g or 2 min. Two microliters of the peptide solution were loaded onto

C-18 (0.075 × 100 mm) nanocolumn coupled to a nanoAcquity ystem. The column was equilibrated with 0.1% FA and eluted with

10% to 85% ACN gradient in 0.1% FA. The eluates were directly nfused into a nanoelectrospray source. The mass spectrometer was perated in positive mode with a source temperature of 373 K and a apillary voltage at 3.0 kV. LC–MS/MS was performed according to he data-dependent acquisition (DDA) method. The lock mass used n acquisition was m/z 785.84 ion of the [Glu1] fibrinopeptide B. he selected precursor ions were fragmented by collision-induced issociation (CID) using argon as the collision gas. All of the CID pectra were manually interpreted and searches for similarity were erformed online using BLAST on the NCBI website.

.11. Artemia lethality test

The Artemia lethality test was conducted according to Carneiro t al. [21]. H-3 and HMA were dissolved in artificial sea water (ASW) t a concentration of 200 g mL−1. The assay was performed board- ng 24-well Linbro® plates in which each well contained 10 Artemia auplii in a final volume of 2 mL. Lectin solution was added to he wells at final concentrations of 12.5, 25, 50 and 100 g mL−1. he experiment was performed in triplicate, and the negative con- rol wells contained 2 mL of artificial seawater with 10 Artemia auplii. After 24 h, the number of dead nauplii in each well was ounted.

.12. Agglutination of bacteria

Escherichia coli and Staphylococcus aureus were grown in nutri-

nt broth at 37 ◦C for 24 h, harvested by centrifugation at 2000 × g or 10 min, washed three times with TBS, suspended in TBS con- aining formaldehyde 4%, kept for 16 h at 4 ◦C, washed three times ith TBS, and, finally, suspended in the same buffer. Bacterial count

ical Macromolecules 72 (2015) 1368–1375

was calculated by the serial dilution method, and A625 was main- tained around 1.0. Bacterial agglutination was tested by mixing 50 L of HMA and H-3 (1 mg mL−1) to an equal volume of bacte- rial suspension. Results were observed under a light microscope after incubation for 2 h [22].

2.13. Antioxidant activity

2.13.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The DPPH assay was conducted according to Blois et al. [23], with some modifications. HMA and H-3 were solubilized in four differ- ent concentrations (12.5, 25, 50 and 100 g mL−1). The absorbance of sample, blank sample, and control was measured at 517 nm, after 30 min incubation in the dark at room temperature, using a Biochrom Asys UVM340 microplate reader (Cambridge, UK). The sample consisted of a mixture of 2.5 mL DPPH methanolic solu- tion (0.16 mM) with 0.5 mL of HMA and H-3. The blank sample consisted of 0.5 mL of HMA and H-3 and 2.5 mL MeOH, while the control contained 3 mL DPPH methanolic solution (0.16 mM) only. The percentage of DPPH scavenging activity was calculated using the expression below:

Scavenging effect (%) = [ 1 −

( Abssample − Abssample blank

) Abscontrol

] × 100%

2.13.2. ˇ-Carotene bleaching assay The method described by Chew et al. [24] was used for the

inhibition of -carotene oxidation. HMA and H-3 were solubilized in four different concentrations (12.5, 25, 50 and 100 g mL−1). Linoleic acid and Tween 40 were added to the -carotene solution in chloroform (100 g mL−1). The chloroform was then evaporated, and oxygen-saturated ultrapure water was added to the residue. The -carotene/linoleic acid emulsion was shaken vigorously, and aliquots of this emulsion were added to HMA and H-3 at different concentrations. The absorbance was read at 470 nm immediately after the emulsion was prepared (Absinitial) and after 1 h of incu- bation in a water bath at 50 ◦C (Abs1h). The percentage AOA was calculated with the following formula:

Antioxidant activity (%) = (

Abs1 h Absinitial

) × 100%

2.13.3. Ferrous ion chelating assay Ferrous ion chelating activity was determined according Wang

et al. [25]. HMA and H-3 were solubilized in four different concen- trations (12.5, 25, 50 and 100 g mL−1). The absorbance of sample, blank sample and control was measured at 562 nm after 10 min incubation at room temperature, using a Biochrom Asys UVM340 microplate reader (Cambridge, UK). The sample consisted of dis-

Ferrous ion chelating activity (%)

= [ Abscontrol −

( Abssample − Abssampleblank

)] Abscontrol

× 100%

R.F. Carneiro et al. / International Journal of Biological Macromolecules 72 (2015) 1368–1375 1371

Table 1 Purification yield of HMA. a Minimal active concentration.

Fraction Titer (HU mL−1) Total protein (mg) Specific activity (HU mg−1) Total activity Yield (%) Purification fold (x) aMAC (g mL−1)



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.1. Purification of HMA-1

A purple lectin was purified from H. manglaris by the combi- ation of hydrophobic interaction chromatography (HIC) and ion xchange chromatography (IEC) (Fig. 1). The lectin was purified .1 times and represents 48% of the total hemagglutinating activity resent in the crude extract (Table 1).

.2. Molecular mass, sugar content and absorbance spectrum

On SDS PAGE, in the presence and absence of reducing agents, MA showed two main bands: one broad band ( chain) and one light band ( chain) with Mr 22,000 and Mr 15,000, respectively Fig. 2A). On native conditions, HMA showed Mr 60,000 on N-PAGE Fig. 2B) and a sharp and symmetrical peak of Mr 55,000 on size xclusion chromatography (Fig. 3). HMA is a glycoprotein with 7% of neutral carbohydrate, as indi-

ated by the phenol-sulfuric acid assay. After testing with PAS, HMA howed positive coloration (Fig. 2C).

Wave scan of HMA showed three absorbance peaks at 216, 80 and 660 nm, indicating the presence of a purple chromophore inked to the lectin (data not shown).

.3. Hemagglutination and inhibition of HMA

HMA was able to agglutinate rabbit erythrocytes, showing arked preference by trypsin-treated erythrocytes, but it could ot agglutinate human erythrocytes (Table 2). Hemagglutinating ctivity was only inhibited by thyroglobulin at minimum inhibitory oncentration (MIC) of 0.5 mg mL−1.

.4. Effects of pH, temperature and divalent cations on the emagglutinating activity of HMA

The hemagglutinating activity of HMA was stable up to 60 ◦C, ith total loss of activity after heating to 80 ◦C. HMA activity was table between pH 8 and 9. The activity decreases at acidic pH and is bolished at pH 4.0 (data not shown). The presence of CaCl2, MnCl2, gCl2 and EDTA did not affect the hemagglutinating activity of MA.

.5. Molecular mass and sulfhydryl groups determination by

ass spectrometry

Reverse phase chromatography (RPC) on C-8 column leads to eparation of HMA chains (Fig. 4). The first peak (-chain) showed

able2 emagglutinating activity of HMA. Hemagglutination was expressed in titer HU mL−1).

Blood type Native Enzymatic treatment

Papain Trypsin

Human A – – – Human B – – – Human O – – – Rabbit – 8 32

7708 100 1 27.89 3648 47 7 3.9

a molecular mass of 14,201 ± 2 Da (Fig. 5A), and the second peak (-chain) showed a molecular mass of 25,262 ± 2 Da (Fig. 5B). Mass differences of 162 Da (hexose) were observed around the -chain value of molecular mass, suggesting the presence of glycoforms.

Interestingly, when the second peak was infused onto mass spectrometer, a strong ionization signal in the low mass zone was observed. This signal corresponded to ion at m/z [M + H]1+ 581.44, most likely because of a weak linkage between chromophore and -chain (Fig. 5C). To identify the structural constituents of the chro- mophore, ion at m/z [M + H]1+ 581.44 was selected and fragmented (Fig. 5D). The fragmentation pattern suggests the presence of dif- ferent chemical groups formed by carbon, nitrogen, oxygen and hydrogen in the chromophore’s structure.

Four half-cysteines were found in the HMA -chain, whereas two half-cysteines were found in the HMA -chain. No free sulfhydryl groups were identified (data not shown).

3.6. N-terminal and amino acid sequencing

N-terminal sequencing analyses of HMA were initially left out because HMA has two polypeptide chains. Thus, after RPC, HMA chains were separately submitted to N-terminal sequencing. The - chain showed a singular N-terminal, APXAPSIENSFQAXV, while the -chain could not be sequenced by Edman degradation, indicating that the N-terminal appears to be blocked.

Tryptic peptides of HMA showed no similarity to any known protein. Table 3 summarizes the amino acid sequences of peptides obtained by LC–MS/MS.

3.7. Artemia lethality test and agglutination of bacteria

In the Artemia lethality test, HMA and H-3 presented low toxicity levels. H-3 killed 7%, 10%, 10% and 13% of the nauplii at concentra- tions of 12.5 g mL−1, 25 g mL−1, 50 g mL−1 and 100 g mL−1. HMA did not kill any Artemia nauplii at the same concentrations.

Neither H-3 nor HMA was able to agglutinate bacterial cells.

3.8. Antioxidant activity

H-3 and HMA showed no capacity to scavenge free radicals, as determined by DPPH. However, both lectins showed a high ability to protect -carotene and moderate the ferrous ion che- lating activity. In the -carotene bleaching assay, H-3 presented 86.69 ± 0.17%, 89.29 ± 0.16%, 91.22 ± 0.14% and 92.54 ± 0.29% of activity at concentrations of 12.5 g mL−1, 25 g mL−1, 50 g mL−1

and 100 g mL−1, respectively, whereas HMA showed 91.07 ± 0.1%, 93.97 ± 0.2%, 94.88 ± 0.08% and 96.71 ± 0.32% of activity at the same respective concentrations (Fig. 6A). The calculated IC50 were 10.569 ± 0.055 g mL−1 and 9 ± 0.037 g mL−1 for H-3 and HMA, respectively. Both activities presented a dose-dependent response.

H-3 presented 12.4 ± 0.2%, 16 ± 0.2%, 17 ± 0.4% and 20.5 ± 0.1% of ferrous ion chelating activity at concentrations of 12.5 g mL−1, 25 g mL−1, 50 g mL−1 and 100 g mL−1, whereas HMA showed 12.4 ± 0.3%, 13.6 ± 0.2%, 20.6 ± 0.2% and 22.5 ± 0.2% of activity at

the same respective concentrations (Fig. 6B). The calculated IC50s were 0.460 ± 0.005 mg mL−1 and 0.322 ± 0.004 mg mL−1 for H-3 and HMA, respectively. Also, both activities occurred in a dose- dependent manner.

1372 R.F. Carneiro et al. / International Journal of Biological Macromolecules 72 (2015) 1368–1375

Fig. 1. Purification of HMA. (A) Approximately 40 mL of crude extract containing (NH4)2SO4 500 mM were applied onto a Phenyl-Sepharose matrix. The column was equilibrated and washed with acetate buffer, pH 5.5, containing (NH4)2SO4 500 mM at a flow rate of 2 mL min−1. Active fractions (P2) were eluted with ethanol 20%, as indicated by arrow. (B) Phenyl Sepharose fraction P2 was dialyzed, freeze-dried and loaded onto a DEAE-Sephacel column equilibrated with Tris buffer, pH 7.6. Active fractions (D1) were termed HMA. Chromatographies were monitored at 280 nm and 660 nm.

Fig. 2. Electrophoresis profile of HMA. (A) SDS PAGE 15%. (M) Molecular marker; (1) Crude extract of H. manglaris; Purified HMA in the absence (2) and presence (3) of 2-mercaptoethanol; (4) Purified H-3. (B) Native PAGE. (1) BSA; (2) Native HMA. (C) SDS PAGE stained with PAS. (1) H-3; (2) HMA.

Fig. 3. Size exclusion profile of HMA in BioSuite 250.5 m HR SEC colunm. Absorbance was monitored at 280 nm and 660 nm.

Table3 Peptides sequenced from the tryptic digests of HMA using tandem mass spectrometry.

Peptide m/z Sequence Mass  (Da)

Observe Calculated

-T1 727.90 QYSGV[L/I]F[L/I]KSGQK 1453.79 1453.79 0.00 -T2 609.72 QNGWDASDPTK 1217.42 1271.53 0.11 -T3 850.30 SCSTTPPQWGFY[L/I]R 1698.58 1698.78 0.20 -T4 597.57 KGNVTGD[L/I]PSFGVGEDAK 1789.70 1789.88 0.18 -T5 646.22 QNGWDDQEATK 1290.44 1290.58 0.14 -T1 539.26 AP[L/I] [L/I]SDSSCK 1076.51 1076.51 0.00 -T2 651.85 [L/I]SD[L/I]DS[L/I] [L/I]AEVK 1301.69 1301.70 0.01 -T3 681.83 P[L/I]A[L/I]SDSSQAFVK 1361.64 1361.71 0.07 -T4 640.23 SSGTSYQ[L/I]PPSR 1278.45 1278.62 0.17 -T5 572.27 FSNYVSW[L/I]K 1142.53 1142.57 0.04

R.F. Carneiro et al. / International Journal of Biological Macromolecules 72 (2015) 1368–1375 1373

Fig.4.ReversephasechromatographyofHMA. Retained fractions were recovered by elution with gradient of ACN (5–90%). The chromatography was monitored at 280 nm.

Fig.5.MolecularmassdeterminationofHMA. (A) Deconvoluted mass spectra of HMA -chain after RPC. (B) Deconvoluted mass spectra of HMA -chain after RPC. (C) Selected ion at m/z [M + H]1+ 581.44. (D) Top-down fragmentation of the ion at m/z [M + H]1+ 581.44. Fragments reveal the presence of organic compounds.

Figure6.AntioxidantactivityofH-3andHMA. (A) -Carotenebleachingassay. H-3 and HMA showed IC50 values of 10.569 ± 0.055 g mL−1 and 9 ± 0.037 g mL−1, respectively. (B) Ferrousionchelatingactivity. H-3 and HMA showed IC50 values of 0.460 ± 0.005 mg mL−1 and 0.322 ± 0.004 mg mL−1, respectively.

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374 R.F. Carneiro et al. / International Journal of


Lectins with natural chromophores could be interesting iotechnological tools. Therefore, having previously reported the urification of H-3, we decided to search for similar lectins. Pro- eins with this characteristic have been studied for their possible tilization as fluorescent probes [26]. For example, the green fluo- escent protein isolated from Aequorea victoria (GFP) has been used n medicine, cell biology and molecular biology.

In another tropical sponge, H. manglaris, we have now found lectin similar to H-3. Termed HMA, this lectin was successfully urified using a combination of HIC and IEC, similar to the purifi- ation of Holothuria scabra [27] and H. caerulea [11]. The high yield btained from this purification process is unusual, but it is similar o that achieved in the purification of H-3 [11].

When exposed to variations of temperature and pH, HMA ehaves in a manner similar to that of other sponge lectins that re often stable up to temperatures of 70 ◦C and in basic-neutral H [28,29]. Basic-neutral pH represents the natural environment of hese animals, and the relative thermal stability of sponge lectins ainly results from the presence of disulfide bonds in their struc-

ure, a common factor in sponge lectins [30,31]. Indeed, MS data evealed the presence of cysteines in HMA. All cysteines found in MA appear be involved in intrachain disulfide bonds. H-3 pre- ented the same amount of cysteines as HMA [11].

HMA is not a metalloprotein, such as lectins isolated from inachyrella and Haliclona species [7,11,29,32]. A few lectins isolated from marine sponges have been shown

o recognize only complex carbohydrates [21,33]. HMA was only nhibited by thyroglobulin, a glycoprotein rich in residues of Glc- Ac, mannose and GalNAc in the terminal position. On the other and, H-3 was inhibited by GalNAc. Although GalNAc did not affect he hemagglutinating activity of HMA, it is possible that HMA ecognizes cores containing GalNAc in the terminal position of yroglobulin.

Like H-3, HMA is composed of two distinct polypeptide chains. In olution, HMA is likely a mixture of two -chains and one -chain, lycosylated or not. Therefore, HMA activity is a heterotrimer, n unusual formation in sponge lectins that typically consist of imers or tetramers linked by weak interactions or disulfide bonds 7,26,34].

The molecular mass of HMA, as determined by MS, is in rea- onable agreement with the mass estimated by electrophoresis. ivergences are sometimes observed between mass determined y MS and mass estimated by SDS-PAGE. These divergences result rom intrinsic characteristics of the protein, such as internal disul- de bonds, glycosylation, or phosphorylation. Eventually, these haracteristics modify the migration of a protein in electrophoresis.

PAS staining and Dubois assay indicated that HMA is a glyco- rotein, and these data were confirmed by MS analysis. Several ponge lectins are glycoproteins [12,28,34]. Although it was possi- le to observe some hexose residues in the -chain, we were not ble to identify the glycan attached to HMA. Unsurprisingly, a molecule of 581 Da was found when the HMA

-chain was analyzed by MS. This molecule is probably a chro- ophore attached to the HMA -chain. Chromophore-581 found

n HMA is an organic molecule much like the phycobilins found n stromata of algal cells. However, unlike phycobilins that are inked to their apoproteins by a thiol ester bond, chromophore- 81 appears to be weakly linked to HMA, as inferred from its easy eparation from HMA without chemical treatment.

H-3 also has a chromophore linked to its -chain by weak

nteractions, but in H-3, the chromophore has a molecular mass f 597 Da, corresponding to one more oxygen molecule that hromophore-581. In phycobilins, a simple modification in chem- cal structure, e.g., the presence/absence of one oxygen molecule

ical Macromolecules 72 (2015) 1368–1375

or translocation of a double bond, can define the coloration of the chromophore [35]. For instance, the structural difference between phycoerythrobilin and phycocyanobilin is the position of a double bond, while the difference between their absorbances is tremendous. Phycoerythrobilin absorbs at 530 nm and emits red coloration, while phycocyanobilin has an absorption peak at 600 nm and emits blue coloration [36]. Thus, it is possible that the absence of a molecule of oxygen in chromophore-581, in compar- ison to chromophore-597, is sufficient to explain the difference of light absorption between the two chromophores (660 nm and 620 nm, respectively).

Although HMA and chromophore-581 being linked through weak interactions, extreme conditions seem be necessary to its separation, i.e. high voltage in mass spectrometry analysis; high concentration of organic solvents. Moderate variations of pH (3–11) and temperature (0–70 ◦C) did not affect interaction, as noted by the incubation of HMA in this conditions and posterior measure- ment by wavescan. Also, -mercaptoethanol, EDTA and urea did not affect interaction (data not shown).

Despite the presence of a chromophore in both H-3 and HMA, they do not share sequence similarity. Moreover, HMA showed no sequence similarity with any protein. Therefore, we were unable to determine the complete sequence of HMA by MS techniques.

Unlike most lectins isolated from marine invertebrates that seem be involved in defense response [22,37,38], neither HMA nor H-3 showed toxic effects against Artemia nauplii. These lectins were also unable to agglutinate bacterial cells. However, their antiox- idant activity, especially on -carotene protection, suggests the putative involvement these lectins in antioxidant protection of the sponges. Interestingly, other sponge lectins have not shown such antioxidant activity by the same methods [39], leading us to believe that this activity is mediated by the chromophores present in HMA and H-3.

Harrison and Cowden [40] suggest that sponge synthesizes pig- ments that act like photoprotectors to minimize the action from solar radiation that could cause damage to vital metabolic products essential to the animal. H. caerulea and H. manglaris are tropical sponges, and in their natural environment, they are constantly assaulted by the physical elements, including high levels of solar radiation. Accordingly, it is possible that lectins or other proteins containing chromophores are involved in photo-oxidation protec- tion.

In conclusion, we have isolated a new lectin from the tropi- cal sponge H. manglaris. Despite sharing some characteristics with H-3, HMA showed no sequence similarity with any protein. Thus, H-3 remains an orphan with respect to the family of lectins. We also observed that lectins containing-chromophores can protect -carotene against oxidation; therefore, we speculate that, in vivo, these lectins could protect sponge metabolites that are essential for survival.


This work was supported by the Brazilian agencies CNPq (Con- selho Nacional de Desenvolvimento Científico e Tecnológico), FUNCAP (Fundaç ão Cearense de Apoio ao Desenvolvimento Cientí- fico e Tecnológico) and FINEP (Financiadora de Estudos e Projetos). The authors thank the National Museum–UFRJ, Rio de Janeiro, Brazil, for the sponge identification. AHS, BSC, CSN and KSN are senior investigator of CNPq.


[1] D.C. Kilpatrick, Handbook of Animal Lectins, Properties and Biomedical Appli- cations, British Library, Eddinburg, 2002.







[ [ [ [





[ [

[ [




[ [



[ [

[ [ [

R.F. Carneiro et al. / International Journal of

[2] G.R. Vasta, H. Ahmed, Animal Lectins: A Functional View, CRC Press, New York, NY, 2008.

[3] G.R. Vasta, Nat. Rev. Microbiol. 7 (2009) 424–438. [4] T. Ogawa, M. Watanabe, T. Naganuma, K. Muramoto, J. Amino Acids. 2011

(2011) 20 (Article ID 838914). [5] R.M. Moura, K.S. Aragão, A.A. Melo, R.F. Carneiro, C.B. Osorio, P.B. Luz, A.F.

Queiroz, E.A. Santos, N.M. Alencar, B.S. Cavada, Fundam. Clin. Pharmacol. 27 (2012) 656–668.

[6] Y. Fujii, N. Dohmae, K. Takio, S.M. Kawsar, R. Matsumoto, I. Hasan, Y. Koide, R.A. Kanaly, H. Yasumitsu, Y. Ogawa, S. Sugawara, M. Hosono, K. Nitta, J. Hamako, T. Matsui, Y. Ozeki, J. Biol. Chem. 287 (2012) 44772–44783.

[7] T. Ueda, Y. Nakamura, C. Smith, B.A. Copits, A. Inoue, T. Ojima, S. Matsunaga, G.T. Swanson, R. Sakai, Glycobiology 4 (2012) 412–425.

[8] X.W. Wang, X.F. Zhao, J.X. Wang, J. Biol. Chem. 289 (2014) 2405–2414. [9] P. Delatorre, B.A.M. Rocha, E.P. Souza, T.M. Oliveira, G.A. Bezerra, F.B.M.B.

Moreno, B.T. Freitas, T. Santi-Gadelha, A.H. Sampaio, W. Azevedo, B.S. Cavada, BMC Struct. Biol. 7 (2007) 52.

10] P. Delatorre, J.S. Silva-Filho, B.A.M. Rocha, T. Santi-Gadelha, R.B. Nóbrega, C.A.A. Gadelha, K.S. Nascimento, C.S. Nagano, A.H. Sampaio, B.S. Cavada, Biochimie 95 (2013) 1–7.

11] R.F. Carneiro, A.A. Melo, A.S. Almeida, R.M. Moura, R.P. Chaves, B.L. Sousa, K.S. Nascimento, S. Saker-Sampaio, J.P.M.S. Lima, B.S. Cavada, C.S. Nagano, A.H. Sampaio, Int. J. Biochem. Cell Biol. 45 (2013) 2864–2873.

12] D. Gundacker, S.P. Leys, H.C. Schröder, I.M. Müller, W.E.G. Müller, Glycobiology 11 (2001) 21–29.

13] H. Kamiya, K. Muramoto, R.R. Goto, Bull. Jpn. Soc. Sci. Fish. 56 (1990) 1159.

14] M. Wiens, S.I. Belikov, O.V. Kaluzhnaya, A. Krasko, H.C. Schroder, S.P. Ottsatadt, W.E.G. Muller, Dev. Genes Evol. 216 (2006) 229–242.

15] M.M. Bradford, Anal. Biochem. 72 (1976) 248–534. 16] A.H. Sampaio, D.J. Rogers, C.J. Barwell, Phytochemistry 48 (1998) 765–769. 17] U.K. Laemmli, Nature 227 (1970) 680–683. 18] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F.F. Smith, Anal. Chem. 28

(1956) 350–356. 19] R.M. Zacharius, T.E. Zell, J.H. Morrison, J.J. Woodlock, Anal. Biochem. 30 (1969)

148–152. 20] A. Shevchenko, H. Tomas, J. Havlis, J.V. Olsen, M. Mann, Nat. Protoc. 1 (2006)




ical Macromolecules 72 (2015) 1368–1375 1375

21] R.F. Carneiro, A.A. Melo, F.E.P. Nascimento, C.A. Simplicio, K.S. Nascimento, B.A.M. Rocha, S. Saker-Sampaio, R.M. Moura, S.S. Mota, B.S. Cavada, C.S. Nagano, A.H. Sampaio, J. Mol. Recognit. 26 (2013) 51–58.

22] A.A. Melo, R.F. Carneiro, W.M. Silva, R.M. Moura, G.C. Silva, O.V. Sousa, J.S. Saboya, K.S. Nascimento, S. Saker-Sampaio, C.S. Nagano, B.S. Cavada, A.H. Sampaio, Int. J. Biol. Macromol. 64 (2014) 435–442.

23] M.S. Blois, Nature 181 (1956) 1199–1200. 24] Y.L. Chew, Y.Y. Lim, M. Omar, K.S. Khoo, LWT—Food Sci. Technol. 41 (2008)

1067–1072. 25] T. Wang, R. Jónsdóttir, G. Ólafsdóttir, Food Chem. 116 (2009) 240–248. 26] P. Bamfield, M.G. Hutchings, Chromic Phenomena: Technological Application

of Colour Chemistry, RSC Publishing, Cambridge, 2010. 27] N.M. Gowda, U. Gowsami, M.I. Khan, Fish Shellfish Immunol. 24 (2008)

450–458. 28] R.M. Moura, A.F.S. Queiroz, J.M.S.L.L. Fook, A.S.F. Dias, N.K. Monteiro, J.K. Ribeiro,

G.E. Moura, L.L. Macedo, E.A. Santos, M.P. Sales, Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 145 (2006) 517–523.

29] D.S. Medeiros, T.L. Medeiros, J.K.C. Ribeiro, N.K.V. Monteiro, L. Migliolo, A.F. Uchoa, I.M. Vasconcelos, A.S. Oliveira, M.P. Sales, E.A. Santos, Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 155 (2010) 211–217.

30] F. Buck, C.I. Luth, H. Bretting, Biochem. Biophys. Acta 1159 (1991) 1–8. 31] F. Buck, C. Schulze, M. Breloer, K. Strupat, H. Bretting, Comp. Biochem. Physiol.

B: Biochem. Mol. Biol. 121 (1998) 153–160. 32] I. Pajic, Z. Kljajic, N. Dogovic, D. Sladic, Z. Juranic, M.J. Gasic, Comp. Biochem.

Physiol. B: Biochem. Mol. Biol. 132 (2002) 213–221. 33] C. Xiong, W. Li, H. Liu, W. Zhang, J. Dou, X. Bai, Y. Du, X. Ma, Comp. Biochem.

Physiol. C: Pharmacol. Toxicol. Endocrinol. 143 (2006) 9–16. 34] P.B. Miarrons, M.M. Fresno, J. Biol. Chem. 275 (2001) 29283–29289. 35] L. Barsanti, P. Gualtieri, Algae: Antamomy, Biochemistry, and Biotechnology,

CRC Press, Boca Raton, FL, 2006. 36] D. Voet, J.G. Voet, Biochemistry, Wiley, New York, NY, 1995. 37] C.A. Janeway, R. Medzhitov, Annu. Rev. Immunol. 20 (2002) 197–216. 38] G.R. Vasta, H. Ahmed, E.W. Odom, Curr. Opin. Struct. Biol. 14 (2004) 617–630.

39] R.R. Dresch, C.B. Lerner, B. Mothes, V.M.T. Trindade, A.T. Henriques, M.M.

Vozari-Hampe, Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 161 (2012) 365–370.

40] F.W. Harrison, R.R. Cowden, Aspects of Sponge Biology, Academic Press, New York, NY, 1976.

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