The International Journal of Biochemistry & Cell Biology 45 (2013) 2864–2873

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H-3, a new lectin from the marine sponge Haliclona caerulea: Purification and mass spectrometric characterization Rômulo Farias Carneiro a , Arthur Alves de Melo b , Alexandra Sampaio de Almeida b , Raniere da Mata Moura c , Renata Pinheiro Chaves b , Bruno Lopes de Sousa a , Kyria Santiago do Nascimento a , Silvana Saker Sampaio b , João Paulo Matos Santos Lima d , Benildo Sousa Cavada a , Celso Shiniti Nagano b , Alexandre Holanda Sampaio b,∗ a Laboratório de Moléculas Biologicamente Ativas – BioMol-Lab, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, s/n bloco 907, 60440-970 Fortaleza, Ceará, Brazil1 b Laboratório de Biotecnologia Marinha – BioMar-Lab, Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Campus do Pici s/n, bloco 871, 60440-970 Fortaleza, Ceará, Brazil2 c Curso de Medicina, Universidade Potiguar, Av. Sen. Salgado Filho 1610, Lagoa Nova, 59054-0 Natal, Rio Grande do Norte, Brazil3 d Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Av. Salgado Filho s/n, Lagoa Nova, 59078-970 Natal, Rio Grande do Norte, Brazil4

a r t i c l e

i n f o

Article history: Received 19 August 2013 Received in revised form 19 September 2013 Accepted 10 October 2013 Available online 19 October 2013 Keywords: Primary structure Chromophore Sponge Lectin Glycosylation

a b s t r a c t A new lectin from the marine sponge Haliclona caerulea (H-3) was isolated using a combination of hydrophobic interaction chromatography and ion-exchange chromatography. H-3 is a protein with three distinct bands on SDS-PAGE: 9 kDa, 16 kDa and 18 kDa. Nevertheless, on gel filtration and N-PAGE, H-3 showed a symmetrical peak and a unique band, respectively. Hemagglutinating activity of H-3 was stable at neutral pH and temperatures up to 60 ◦ C. N-Acetylgalactosamine and porcine stomach mucin were the most potent inhibitors of H-3. Primary structure of the lectin was determined using tandem mass spectrometry, and it showed no similarity to any members of the animal lectin families. Top down fragmentation revealed some posttranslational modifications in H-3, including glycosylation. The glycan composition of H-3 was determined, and its structure was predicted. Furthermore, H-3 is a blue protein, binding to a chromophore(-597) by weak interactions, and this is the first time that the interaction between one lectin and a natural chromophore has been shown. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Since the original work of Dodd et al. (1968), lectins from marine sponges have been isolated, characterized, and, to some extent, their biological roles have been established, in contrast to other marine invertebrate lectins. For instance, a galectin isolated from Geodia cydonium interacted with aggregation factors and precipitates of the reaggregation process (Muller, 1997). Two lectins isolated from Apphrocallistus vastus are also involved in the reaggregation process (Gundacker et al., 2001), and in Halichondria panicea,

∗ Corresponding author at: BioMar-Lab, Departamento de Engenharia de Pesca, Universidade Federal do Ceara, Av. Mister Hull, 60440-970, Box 6043, Fortaleza, Ceara, Brazil. Tel.: +55 85 33669728; fax: +55 85 33669728. E-mail address: [email protected] (A.H. Sampaio). 1 Tel.: +55 85 33669818. 2 Tel.: +55 85 33669728. 3 Tel.: +55 84 32151290. 4 Tel.: +55 84 32153416. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.10.005

a d-galacturonic acid binding-lectin seems to be involved in the maintenance of symbiosis (Muller et al., 1981). In recent years, lectins isolated from sponges have attracted extensive attention because of their biological properties, such as toxic effects against malignant cells (Rabelo et al., 2012), modulatory activity in the mammalian nervous system (Ueda et al., 2012) and proinflammatory effects (Queiroz et al., 2008). However, structural features of those lectins are understudied. In a pioneering work, two lectins from Axinella polypoides (lectin I and II) were sequenced by Edman degradation and mass spectrometry (Buck et al., 1991, 1998). Axinella lectins shared 65% of identity among themselves, but show no identity with other lectins (Buck et al., 1991). In another study, Pfeifer and coworkers deduced the amino acid sequence of two isolectins from the siliceous sponge G. cydonium by cDNA cloning. LECT-1 and LECT-2 showed a sequence of 38 conserved amino acids, which is characteristic of the carbohydratebinding site of vertebrate galectins (Pfeifer et al., 1993). Then, the amino acid sequence of two isolectins from A. vastus was

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deduced by cDNA cloning (Gundacker et al., 2001). APHR-LECC1 and APHR-LECC2 were classified as C-type lectins. Aphrocallistes lectins showed highest similarity to type-II membrane proteins from higher metazoan phyla (Gundancker et al., 2001). Recently, the amino acid sequence of CchG-1, a galectin isolated form Cinachyrella sp. (ball sponge), was deduced by cDNA cloning (Ueda et al., 2012), and for the first time, the three-dimensional structure of a sponge lectin was determined by X-ray crystallography (Freymann et al., 2012). CchG-1 adopts a novel tetrameric arrangement in which a rigid toroidal-shaped ‘donut’ is stabilized in part by the packing of pairs of vicinal disulfide bonds (Freymann et al., 2012). In a previous study, we isolated two lectins from the Caribbean sponge Haliclona caerulea: H-1 and H-2. H-1 is a monomeric protein with relative mass of 40 kDa, whereas H-2, which is a dimeric protein with relative mass of 15 kDa, recognizes O-linked glycoproteins. H-2 had its primary structure partially determined by tandem mass spectrometry (MS/MS), but it showed no similarity to any of the known lectins (Carneiro et al., 2013). Here, we report the purification, characterization, glycan composition and amino acid sequence of H-3, a new lectin from the marine sponge H. caerulea. Also, we identified one putative lectin in the genome of the sponge Amphimedon queenslandica. 2. Materials and methods 2.1. Animal collection Specimens of the marine sponge H. caerulea were collected in the intertidal zone of Paracuru Beach, Paracuru, Ceará State, Brazil. Fresh sponges were transported on ice to the laboratory and stored at −20 ◦ C until use. 2.2. Isolation of H-3 Frozen sponges were cut into small pieces, triturated into powder and extracted (1:2, w/v) with 20 mM acetate buffer pH 5.5 (AB). The mixture was strained through nylon tissue and clarified by centrifugation for 20 min at 10,000 × g at 4 ◦ C. The supernatant (crude extract) was collected and assayed for hemagglutinating activity and protein concentration (Bradford, 1976). (NH4 )2 SO4 was added to crude extract to achieve the final concentration of 500 mM, and the suspension was left for 4 h at 4 ◦ C. The precipitated proteins were removed by centrifugation for 20 min at 10,000 × g at 4 ◦ C, and the supernatant (H-3-enriched fraction) was loaded on a Phenyl-Sepharose 6B column (1.0 cm × 5.0 cm) equilibrated with 500 mM of (NH4 )2 SO4 in AB. The column was washed with the same buffer at a flow rate of 1 mL min−1 until the column effluents showed absorbance of less than 0.02 at 280 nm. Two adsorbed fractions (P1 and P2 ) were eluted with 200 mM (NH4 )2 SO4 in AB and AB alone, respectively. The chromatography was monitored at 280 nm and 620 nm, and 3-mL fractions were manually collected and tested for hemagglutinating activity. The active fraction (P2 ) was dialyzed against deionized water, freeze-dried, solubilized in a small volume of 20 mM Tris–HCl buffer, pH 7.6 (TB), and loaded onto a DEAE-Sephacel column (1.0 cm × 3.0 cm) previously equilibrated with TB. The flow rate was adjusted to 1 mL min−1 , and the column was washed with TB, followed by elution of two adsorbed fractions (D1 and D2 ) with 100 mM and 1 M of NaCl in TB. The chromatography was monitored at 280 nm and 620 nm, and 1-mL fractions were collected. Fraction D1 corresponded to pure Halilectin 3 (H-3).

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2.3. Hemagglutinating activity and hemagglutination inhibitory assay The hemagglutination tests were performed in microtiter plates with V-bottom wells using the two-fold serial dilution method. One hemagglutinating unit (HU) was defined as the amount of lectin able to agglutinate and, hence, precipitate the erythrocytes in a suspension 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 (Sampaio et al., 1998). The following carbohydrates and glycoproteins were used: d-fructose, d-fucose, dgalactose, d-glucose, d-mannose, methyl-␣-d-galactopyranoside, methyl-␣-d-glucopyranoside, N-acetyl-d-galactosamine, N-acetyld-glucosamine, N-acetyl-d-mannosamine, d-sucrose, ␣-lactose, ␤-lactose, lactulose, carragenan, fucoidan, tyroglobulin, ovomucoid and porcine stomach mucin (PSM). The initial concentrations of the inhibitors were 100 mM for sugars and 2 mg mL−1 for glycoproteins. Light microscopy was used to evaluate both hemagglutinating activity and the hemagglutination inhibition assay. 2.4. Physicochemical properties of H-3 Homogeneity of the native H-3 was evaluated by N-PAGE 15%. Molecular mass of H-3 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 (1970). LMW-SDS Marker kit (GE) and BSA were used as the standard. The relative mass of the native H-3 was estimated by gel filtration on Sephacryl S-200 HR 16/60 (1.6 cm × 60 cm), equilibrated with 50 mM Tris–HCl, pH 8.0, containing NaCl 500 mM. The column was 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 H-3 was evaluated, as described by Dubois et al. (1956), using lactose as the standard. Glycoproteins in SDS-PAGE were stained with periodic acid-Schiff (PAS), as described by Zacharius et al. (1969). 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 H-3. 2.5. Effects of pH, temperature and divalent cations on the hemagglutinating activity of H-3 The effects of pH, temperature, EDTA and divalent cations on lectin activity were evaluated as described by Sampaio et al. (1998). 2.6. Reverse phase and separation of H-3 chains Purified H-3 was solubilized with 5% acetonitrile (ACN) containing 0.1% formic acid (FA) and submitted to reverse phase chromatography (RPC) coupled to the Pharmacia Biotech AKTA Purifier 10 System. Sephasil Peptide C-8 10/250 column (Amersham) was equilibrated and washed with ACN 5% containing FA 0.1% at a flow rate of 1 mL min−1 . Retained proteins were recovered by two elution steps. First, a gradient of 5%–70% of ACN containing FA 0.1% was performed. Afterwards, retained fractions were recovered by elution with ACN 90%. Absorbance of the column eluate was monitored at 216, 280 and 620 nm, and fractions of 1 mL were collected.

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2.7. Liquid chromatography and mass spectrometry of intact protein LC–MS analyses were performed using a hybrid Synapt HDMS mass spectrometer (Waters Corp., Milford, MA, USA). The instrument was calibrated with [Glu1]fibrinopeptideB fragments. Mass spectra were acquired by scanning at m/z range from 500 to 3000 at 1 scan/s. Two ␮g of intact H-3 were applied to a C-18 nanocolumn and eluted with gradient of 10% to 85% of ACN containing FA 0.1%. Eluates were directly injected into the mass spectrometer using a nanoACQUITY UPLC System (Waters Corp.) connected to a NanoElectrospray Mass Spectrometry source. The mass spectrometer was operated in positive mode, using a source temperature of 363 K and capillary voltage at 3.5 kV. Data collection and processing were controlled by Mass Lynx 4.1 software (Waters). Deconvolution of ESI mass spectra was performed using the MaxEnt 1 algorithm in the Mass Lynx software. Indeed, retained fractions in RPC-AKTA were directly infused onto the mass spectrometer. The instrument was operated as described above. To determine H-3 native average mass, the lectin was solubilized in 25 mM ammonium bicarbonate and directly infused into the mass spectrometer. The mass spectrometer was operated in positive mode, using a source temperature of 323 K and capillary voltage at 2.0 kV. 2.8. Quantification of sulfhydryl groups For quantization of free cysteine residues in H-3, the lectin was incubated with iodoacetamide 55 mM (IAA) for 45 min at room temperature, subjected to desalting by RPC on the C-8 Sephasil column and analyzed by mass spectrometry as described above. For quantization of total cysteine residues in H-3, the lectin was reduced with dithiothreitol (DTT) 10 mM at 56 ◦ C for 1 h and alkylated with IAA 55 mM in the dark for 45 min at room temperature. Carboxyamidomethylated H-3 was subjected to desalting by RPC on a C-8 Sephasil column and analyzed by mass spectrometry as described above. 2.9. Identification of posttranslational modifications (PTMs) by mass spectrometry Intact H-3 was solubilized in FA 0.1% containing ACN 50%. After centrifugation, H-3 solution was directly injected into a nanoelectrospray source using a Hampton syringe. The flow rate was maintained at 1 ␮L min−1 ; capillary voltage and the cone voltage were set at 3 kV and 40 V, respectively. The source temperature was maintained at 373 K, and nitrogen was used as a drying gas (flow rate of 150 L h−1 ). For Top Down fragmentation, various mass-to-charge ratios were selected using a 1 m/z unit ion isolation window, and selected ions were fragmented by collisions using 20% relative collision energy. 2.10. Chemical deglycosylation of H-3 H-3 (1 mg mL−1 ) was solubilized in NaOH 88 mM containing NaBH4 1 M and maintained at 60 ◦ C for 48 h. After incubation, deglycosylated H-3 was recovered by precipitation with cold acetone. The pellet was solubilized in FA 0.1% and submitted to LC–MS analysis as described above. 2.11. Primary structure determination by tandem mass spectrometry (MS/MS) SDS-PAGE was performed as described above. Gels used for digestion with Asp-N and Glu-C were supplemented with ethylene

glycol diacrylate (EDA) as cross-linker, to a final concentration of 0.22%. EDA plugs were removed with NH4 OH treatment conforming to the description of Bornemann et al. (2010). After staining, H-3 spots were excised, reduced with DTT and carboxyamidomethylated with IAA as described by Shevchenko et al. (2006). Treated spots were subjected to digestion with the following enzymes: trypsin, chymotrypsin, Glu-C, Asp-N, thermolysin and pepsin. Digestion with trypsin and chymotrypsin was performed in ammonium bicarbonate 25 mM at 1:50 (w/w) (enzyme/substrate). Digestion with Glu-C and Asp-N was performed in ammonium bicarbonate 25 mM at 1:200 (w/w) (enzyme/substrate). Pepsin digestion was performed in HCl 0.1 M at 1:50 (w/w) (enzyme/substrate). All digestions were maintained at 37 ◦ C for 18 h. The digestion was stopped with 2 ␮L of 2% FA. The samples were washed four times with 5% FA in 50% ACN. The supernatants were collected and transferred to fresh tubes, pooled, vacuum-dried, solubilized in 20 ␮L of 0.1% FA, and centrifuged at 10,000 × g for 2 min. Two microliters of the peptide solution were loaded onto a C-18 (0.075 mm × 100 mm) nanocolumn coupled to a nanoAcquity system. The column was equilibrated with 0.1% FA and eluted with a 10%–85% ACN gradient in 0.1% FA. The eluates were directly infused into a nanoelectrospray source. The mass spectrometer was operated in positive mode with a source temperature of 373 K and a capillary voltage at 3.0 kV. LC–MS/MS was performed according to the data-dependent acquisition (DDA) method. The lock mass used in acquisition was m/z 785.84 ion of the [Glu1] fibrinopeptide B. The selected precursor ions were fragmented by collision-induced dissociation (CID) using argon as collision gas. All of the CID spectra were manually interpreted. 2.12. Bioinformatics analysis The sequence similarity of the peptides was evaluated online (http://www.ncbi.nlm.nih.gov/BLAST/) using the tBLASTn program with the database whole-genome shotgun contigs (wgs) available from the National Center for Biotechnology Information (NCBI). Contig7463 of the A. queenslandica genome (ACUQ00000000) exhibited similarity with H-3 fragments. Therefore, contig7463 was treated with GenMark (Lonsadze et al., 2005) using a training set from the genome of C. elegans for exonic identification. The alignment between the predicted mRNAs from contig7463 and the genomic contig was performed using the Spidey alignment program (http://www.ncbi.nlm.nih.gov/spidey/spideyweb.cgi). The alignment of the sequences of the putative protein from A. queenslandica and the sequenced peptides of H-3 was performed using ESPript2.2 (Gouet et al., 2003). 3. Results 3.1. Purification of Halilectin 3, the blue lectin of H. caerulea The crude extract from H. caerulea showed lectin activity against human and rabbit erythrocytes. The successive purification by (NH4 )2 SO4 precipitation, phenyl-sepharose 6B (Fig. 1A) and DEAESephacel (Fig. 1B) results in a pure protein. Purification steps result in a recovery of 21%, and purity was increased by 8-fold (Table 1). 3.2. Physical chemical properties of Halilectin 3 SDS-PAGE, in the presence or absence of ␤-mercaptoethanol, revealed three bands with relative mass of the 18, 16 and 9 kDa (Fig. 2A). H-3 appeared as a single band in N-PAGE (Fig. 2B). On gel filtration, H-3 is homogeneous, showing a relative mass of 40 kDa (Fig. 3).

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Table 1 Purification yield of H-3. Fraction

Protein (mg mL−1 )

Specific activity (UH mg−1 )

Total protein (mg)

Purification (-fold)

Recovery in protein (%)

Crude extract H-3-enriched fraction Phenyl DEAE

0.451 0.172 0.382 0.056

70.953 186.046 375.078 571.428

25.256 10.320 2.292 0.784

1 2.62 5.28 8.05

100 40.86 9.08 3.10

The phenol-sulfuric acid assay indicated that H-3 is a glycoprotein with 2.5% of neutral carbohydrate contents. Staining with PAS confirmed the glycoprotein nature of H-3 (Fig. 2C). Wave scan of H-3 showed three absorbance peaks: 216, 280 and 620 nm, suggesting a blue chromophore linked to the lectin (data not shown). 3.3. Hemagglutination assays and inhibition by sugars and glycoproteins Among the tested erythrocytes, H-3 most effectively agglutinated trypsinized human blood group B and A erythrocytes, but was not able to agglutinate blood group O and rabbit erythrocytes. The carbohydrate binding specificity of H-3 was determined by the inhibition of the hemagglutinating activity by sugars and glycoproteins. Among the substances tested, N-acetyl-galactosamine (12.5 mM) and PSM (0.165 mg mL−1 ) were the only effective inhibitors of H-3 hemagglutination (Table 2). Fig. 4 show H-3 hemagglutinating activity and galNAc inhibitory effect.

Fig. 1. Purification of H-3 by combining hydrophobic interaction chromatography and ion exchange chromatography. (A) Approximately 40 mL of H-3 enriched fraction were applied onto a phenyl-sepharose column equilibrated with acetate buffer pH 5.5 containing (NH4 )2 SO4 500 mM. The column was washed with the same buffer at a flow rate of 1 mL min−1 , and two adsorbed fractions (P1 and P2 ) were eluted with 200 mM and 0 mM of (NH4 )2 SO4 in the acetate buffer. (B) Phenyl sepharose fraction P2 was dialyzed and loaded onto a DEAE-sephacel column equilibrated with Tris buffer pH 7.6. Active fractions (D2 ) were named H-3. Chromatographies were monitored at 280 nm and 620 nm.

3.4. Effects of pH, temperature and divalent cations on the hemagglutinating activity of H-3 Hemagglutinating activity of H-3 was maximal between pH 6.0 and 7.0, but outside these values, hemagglutinating capacity was reduced, and the lectin was inactivated at pH 4.0. H-3 was stable up to a temperature of 60 ◦ C, with total loss of activity after heating at 70 ◦ C. Dialysis against EDTA or inclusion of divalent cations, such as Ca2+ , Mg2+ and Mn2+ , did not affect lectin activity. 3.5. Reverse phase chromatography RPC revealed three distinct peaks (RP-I, RP-II and RP-III) with maximum absorption at 280 nm (Fig. 5A). On SDS-PAGE, RP-I was a unique band with 9 kDa (␤-chain), RP-II was a broad band with 16–18 kDa (␣ and ␣ chains), and RP-III could not be observed

Fig. 2. Electrophoresis profile of H-3. (A) Purified H-3 in the absence and presence of 2-mercaptoethanol (lanes 1 and 2). (B) Native H-3 (lane 4) and BSA (lane 3). (C) Lectin from Luetzelburgia auriculata, a glycoprotein (lane 5) and purified H-3 stained with PAS (lane 6). M, molecular markers.

Fig. 3. Estimation of the molecular mass of H-3 by gel filtration on S200. The column was equilibrated with Tris buffer 50 mM, pH 8.0, containing NaCl 500 mM and calibrated with (1) BSA, 66 kDa; (2) ovalbumin, 45 kDa; (3) carbonic anhydrase; 29 kDa; (4) ribonuclease A, 14 kDa and (5) aprotinin, 7 kDa.

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Fig. 4. Hemagglutinating activity of H-3. (A) Hemagglutination of A type erythrocytes treated with trypsin in the presence of H-3. (B) H-3 inhibited by galNac 50 mM. (C) Control group: erythrocytes in saline solution.

Fig. 5. Isolation of H-3 chain by reverse phase chromatography. The column was equilibrated with FA 0.1% containing ACN 5%. Retained fractions were recovered by elution with linear gradient of ACN (5–70%) and one final step of ACN 90%. The chromatography was monitored at 216 nm, 280 nm and 620 nm. Inserted: SDS-PAGE profile of retained fractions in RPC: purified H-3 (lane 1); RP-II (lane 2); RP-I (lane 3) and RP-III (lane 4). M, molecular markers.

(Fig. 5B). RP-III (chromophore) did not absorb at 216 nm or 620 nm, but it did show an orange coloration (data not shown). 3.6. Determination of molecular mass of H-3 LC–MS of intact H-3 revealed two peaks. The first peak (␤-chain) showed molecular mass of 11,828 ± 2 Da after MS analysis (Fig. 6A). The second peak was a mixture of two distinct specimens with molecular mass of 18,290 ±2 Da (␣-chain) and 20,393 ± 2 Da (␣ chain) (Fig. 6B). Table 2 Inhibitory effects of sugars and glycoprotein in hemagglutinating activity of H-3. The initial concentrations of the inhibitors were 100 mM for sugars and 2 mg mL−1 for glycoproteins. NI: no inhibition. Sugars

H-3 (MICa )

d-Galactose d-Glucose d-Mannose d-Fucose Methyl-␣-d-galactopiranoside Methyl-␣-d-glucopiranoside d-GalNAc d-GlcNAc d-ManNAc d-Sucrose ␣-d-Lactose ␤-d-Lactose d-Lactulose d-Fructose Carragenan Fucoidan Glycoproteins PSM Tyroglobulin Ovomucoid

NI NI NI NI NI NI 12.5 mM NI NI NI NI NI NI NI 2.5 mg mL−1 NI

a

Minimal inhibitory concentration.

0.165 mg mL−1 NI NI

Small differences were observed around ␣- and ␤-chain values of molecular mass, suggesting the presence of isoforms and/or adduct formations. Mass spectrum of the ␣ -chain indicates the presence of successive additional ions with mass differences of 162 Da (one hexose residue), suggesting the presence of glycoforms in the ␣ -chain (Fig. 6C). When RPC fractions (RP-I, RP-II and RP-III) were infused in mass spectrometer, the results were similar. RP-I showed a molecular mass identical to that of ␤-chain, and RP-II showed molecular mass identical to that of ␣ and ␣ chains (data not shown). Moreover, RPIII showed molecular mass of 597 Da, and as a result, it was named chromophore-597 (Fig. 6D). 3.7. Determination of sulfhydryl groups Four half-cysteines were found in the H-3 ␣-chain, whereas two half-cysteines were found in the H-3 ␤-chain. No free sulfhydryl groups were identified (data not shown). 3.8. Identification of PTMs by MS analysis Direct infusion of intact H-3 showed only two ion series related to the ␣ and ␣ chains (Fig. 7). The precursor ion [M+12H]12+ at m/z 1700.9219 (␣ chain-related ion) was filtered and fragmented. The resultant ion at m/z 1663.4371 was a [M+12H]12+ ␣ chainrelated ion, suggesting that the ␣-chain and ␣ -chain are identical polypeptide chains and that a PTM of 2103 Da separates them. Therefore, ␣-chain and ␣ -chain show differences in their glycosylation states. Thus, these data suggest a conformation that includes two ␤chains (2× 11 kDa) and either one ␣-chain (18 kDa) or one ␣ -chain (20 kDa), totaling 40 kDa and 42 kDa for H-3 native state, which are approximate values for the mass measurement by gel filtration and PAGE.

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Fig. 6. Molecular mass determination of H-3. (A) Deconvoluted mass spectra of H-3 ␣-chain after LC–MS. (B) Deconvoluted mass spectra of H-3 ␤-chain after LC–MS. (C) Zoom in deconvoluted mass spectra of H-3 ␤-chain after LC–MS. (D) Mass spectra of chromophore-597 purified by RPC (RP-III).

When intact H-3 was directly infused in mass spectrometer and capillary voltage and source temperature were maintained at 2.0 kV and 50 ◦ C, respectively, two different ion series were observed. Maximization of entropy indicated that these two ion series corresponded to ␣-chain and that ␣-chain increased by 596 Da, with molecular mass of 18,294 ± 2 Da and 18,890 ± 2 Da, respectively (Fig. 8). The precursor ion [M+7H]7+ at m/z 2699.7153 (␣ increased-related ion) was filtered and fragmented. The resultant ions [M+7H]7+ at m/z 2614.4539 and [M+1H]1+ at m/z 597.50 ± 2 Da were an ␣-chain-related ion and chromophore-597, respectively (Fig. 9), suggesting that the ␣-chain interacts with chromophore597. 3.9. Deglycosylation of H-3 H-3 was successfully deglycosylated using chemical methods. However, deglycosylated lectin did not show the molecular mass provided by the Top Down experiment (18,290 ± 2 Da). Instead, deglycosylated H-3 showed a molecular mass of 17,548 ± 2 Da, suggesting that the ␣-chain is glycosylated (Fig. 10). The difference in molecular mass between ␣ -chain (20,392 ± 2 Da) and deglycosylated H-3 (17,548 ± 2 Da) was 2862 Da, corresponding to seven units of hexose, seven Nacetylhexosamine and two deoxyhexose residues (Hex7 HexNAc7 DeoxyHex2 ). Furthermore, the presence of different glycan compositions was observed in the ␣-chain of H-3. The ␣-chain may receive from one to six additional hexose residues in the terminal portion, reaching to a maximum glycosylation composition of Hex13 HexNAc7 DeoxyHex2 . 3.10. Primary structure of H-3 Tandem MS data were collected, and peptide sequences were determined via de novo sequencing. The peptides from the different enzymatic digests produced overlapping data that were aligned to create the primary sequence of H-3 chains (Fig. 11).

Overall, approximately 92% of the ␣-chain was sequenced and found to consist of 145 residues, totaling 16,245 Da; however, this value is lower than that determined for deglycosylated lectin (17,548 ± 2 Da). Since the N-terminus of the ␣-chain was found to be blocked by the presence of a pyroglutamic acid residue, missing amino acids must be at the C-terminal. On the other hand, the amino acid sequence was completely determined for the ␤-chain. The ␤-chain consists of 106 residues, of which only two could not be identified by MS/MS. However, they were identified by its similarity with one putative protein of A. queenslandica: pAqp (see below). The 106 residues together totaled 11,622 Da, which is 206 Da lower than the molecular mass determined by MS (11,828 ± 2 Da). This difference could be explained by the presence of an N-glycosylation site containing one Nacetylhexosamine residue (203 Da) in the T-3 peptide, whereas T-3 showed a difference of 203 Da between its determined and calculated molecular masses. Sequence heterogeneities were observed in both sequences. The ␣-chain showed heterogeneity in three positions: 28 (A/V), 35 (G/S) and 38 (S/D). Five positions of heterogeneity were observed in the ␤-chain: 27 (D/E), 49 (T/L), 59 (K/R), 94 (S/D) and 100 (L/Y). Each chain also has one N-glycosylation site: 73 NET76 in the ␣-chain and 65 NTL67 in the ␤-chain. H-3 chains showed no similarity to any known lectin, but one putative protein encoded on the genome of A. queensalndica (pAqP) was identified and showed high similarity to H-3. pAqP was identified by a search of H-3 peptides using the tBLASTn program with database whole-genome shotgun contigs (wgs). Two distinct polypeptide chains were identified. ␣pAqP is composed of 197 amino acids containing one N-glycosylation site and a signal peptide of 19 residues. ␤pAqP is composed of 187 amino acids containing one N-glycosylation site and a signal peptide of 18 residues. Alignments between pAqP and H-3 were realized and revealed identity of 89% to ␣-chains and 88% to ␤-chains (Fig. 12).

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Fig. 7. Top down fragmentation of ␣-chain of H-3 (RP-II). (A) Mass spectra of RPII, using nano ESI infusion at 1 ␮L min−1 . (B) Selection of one ␣ -chain-related ion [M+12H]12+ at m/z 1700.9219. (C) Fragmentation of selected ion by 20% of relative collision energy showed one ␣-chain-related ion [M+12H]12+ at m/z 1663.4371.

4. Discussion Marine sponges represent a rich and still under explored source of new lectins. Currently, about forty lectins have been isolated from sponges. In contrast with other lectins isolated from marine

Fig. 8. Deconvoluted mass spectra of H-3 ␣-chain in the presence of ammonium bicarbonate. NanoESI infusion 1 ␮L min−1 of H-3 10 pmol in ammonium bicarbonate was used. Source temperature and capillary voltage were maintained at 50 ◦ C and 2 kV, respectively.

Fig. 9. Top down fragmentation of ␣-chain native conformation. (A) Mass spectra of RP-II, using nano ESI infusion 1 ␮L min−1 and ammonium bicarbonate as solvent. Source temperature and capillary voltage were maintained at 50 ◦ C and 2 kV, respectively. (B) Selection of one ␣-chain-increased-related ion [M+7H]7+ at m/z 2699.7153. (C) Fragmentation of selected ion by 20% of relative collision energy showed one ␣-chain-related ion [M+7H]7+ at m/z 2614.4539 and chromophore-597 ion [M+1H]1+ at m/z 597.50 ± 2 Da.

invertebrates, lectins from sponges have known physiological roles. Sponge lectins seem to be involved in the process of cellular aggregation (Gundacker et al., 2001), symbiosis (Muller et al., 1981), as well as defense and growth regulation (Wiens et al., 2006). Nevertheless, these lectins lack structural information. Therefore, in this work, we discussed the isolation, amino acid sequence and some biochemical properties of a GalNAc-binding blue lectin from

Fig. 10. Deglycosylated H-3 ␣-chain. H-3 was chemically deglycosylated using NaOH and NaBH4 . Glycan composition was determined by subtraction of the mass values of the partially glycosylated forms of H-3. hexose (); oxidized hexose (); deoxyhexose (); hexosamine ().

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Fig. 11. Primary structure of H-3. (A) ␣-Chain; (B) ␤-chain. Amino acid sequence of H-3 assembled from sequences of overlapping degradation products generated by cleavage with trypsin (-T-), chymotrypsin (-Q-), Glu-C (-G-), Asp-N (-A-), pepsin (-P-) and thermolysin (-Th-). Glycosylation sites are underscored, and sequence heterogeneities of variable positions are marked by (*).

the marine sponge H. caerulea named H-3. The new lectin was purified by a combination of hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC). HIC and IEC have been shown to be a very useful step for the purification of lectins from marine organisms, such as marine algae (Ainouz et al., 1995), and invertebrates (Gowda et al., 2008; Moura et al., 2012; Xiong et al., 2006). After performing HIC-IEC, the new lectin from H. caerulea was found to present 3% of total soluble proteins, an unusual value for recovery of sponge lectins. Values of recovery as high as this have thus far only been seen in Halichondria okadai: HOL-I = 2.7% and HOL-II = 2.9% (Kawagishi et al., 1994). H-3 was shown to be specific toward human blood groups A and B enzyme-treated, and hemagglutination was inhibited only by PSM and GalNAc, but not galactose. The specific recognition of GalNAc suggests the involvement of the N-acetyl group in the binding process. Sponge lectins usually recognize galactose and GalNAc (Gundacker et al., 2001; Moura et al., 2006; Ueda et al., 2012), but just one marine sponge lectin was reported to recognize GalNAc and not galactose: HoL-I, isolated from Halichondria okadai (Kawagishi et al., 1994). On the other hand, sponge lectins that recognize O-linked glycoproteins were described in Craniella australiensis (Xiong et al., 2006), Aplysina archeri and A. lawnosa (Miarrons; Fresno, 2000), and H. caerulea (Carneiro et al., 2013). Porcine stomach mucin (PSM) is a glycoprotein rich in galactose and GalNAc residues. In addition, it contains branches terminated with fucose and sialic acid. The recognition of PSM by H-3 can be explained by the ability of H-3 to recognize GalNAc.

Hemagglutinating activity of H-3 was independent of the presence of divalent cations. Recently, various cation-independent lectins have been isolated from marine sponges, such as H-1 and H-2, ACL-I and ACL-II (lectins isolated from Axinella corrugata) and HOL-III (isolated from Halichondria okadai) (Carneiro et al., 2013; Dresch et al., 2008; Kawsar et al., 2008). Top down fragmentation, N-PAGE and gel filtration suggest that H-3 quaternary structure is organized in a heterotrimer. Several quaternary arrangements have been reported in sponge lectins. Dimeric (Pajic et al., 2002), trimeric (Xiong et al., 2006), tetrameric (Miarrons and Fresno, 2000) and multimeric (Medeiros et al., 2010) proteins linked by weak interactions or disulfide bonds were described, but no sponge lectin has native arrangement similar to H-3. Slight differences were observed between H-3 relative mass calculated by SDS-PAGE and H-3 molecular mass obtained by MS analysis. Lectins isolated from the marine algae Hypnea cervicornis (HCA) and H. musciformis (HML) also show differences between their relative molecular masses as calculated by SDS-PAGE and as determined by MS analysis (Nagano et al., 2005). On SDS-PAGE, the migration of proteins can be influenced by the hydrodynamism and compaction of the molecule. HML and HCA present seven disulfide bonds, making these molecules very compact and leading to a fast migration on SDS-PAGE, creating a divergence between the molecular mass measured by this technique and MS. This slight difference between molecular masses of H-3 could be explained by the presence of intramolecular disulfide bonds. Furthermore, internal disulfide bonds are important structural

Fig. 12. Alignment of H-3 and a putative protein of Amphimedon queensalandica. (A) ␣-Chain alignment; (B) ␤-chain alignment.

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features in the stabilization of the tridimensional structure of proteins. They stabilize proteins by reducing the entropy of the denatured state (Betz, 1993). Thus, the relative thermo- and pH-stability of H-3 could result from the presence of internal disulfide bonds. The phenol-sulfuric acid assay and coloration with PAS confirmed the glycosylation of H-3. To determine glycan composition and to measure deglycosylated molecular mass, chemical deglycosylation and Top Down fragmentation were performed. Fortunately, after chemical deglycosylation, the result was a mixture of partially glycosylated and deglycosylated forms; thus, it was possible to determine the glycan composition by subtracting the mass values of consecutive ions. We were, however, unable to determine the glycan structure of H-3 by mass spectrometry, but the prediction of glycan could be performed using data available in the literature. Several sponge lectins were defined as glycoproteins, including the lectins isolated from Cliona varians (Moura et al., 2006), Aphrocallistes vastus (Gundacker et al., 2001), Aplysna lawnosa, A. archeri (Miarrons and Fresno, 2000) and Axinella corrugata (Dresch et al., 2008), but none of these had their glycidic composition determined. H-3 is a blue protein because it interacts with chromophore597. Chromophore-597 does not absorb at 216 nm, but at 280 nm, indicating the presence of aromatic ring groups. The interaction between H-3 and chromophore-597 characterizes H-3 as a pigment-protein. Pigment-proteins have been studied in marine algae. These proteins (phycobiliproteins) are found in stromi of algae cells and are involved in light capitation for photosynthesis (Glazer, 1982; Maccoll, 1998). The pigment portions of pigmentproteins are known as phycobilins (Wedeimayer et al., 1992). The phycobilins consist of an open chain of four pyrrole rings. Like H-3 pigment, phycobilins are able to absorb light in determined wavelengths, including UV radiation and visible spectrum. However, unlike phycobilins that interact with proteins by thiol-ester bond, chromophore-597 is linked to H-3 by weak interactions. Interestingly, after the disruption of the interaction, chromophore597 presents orange coloration, showing maximum absorption at 450 nm, suggesting some kind of structural modification in its native conformation. We could not determine the structure of chromophore-597, but its light absorption characteristics and its molecular mass lead us to believe that it is a molecule similar to phycobilins. Lectins are proteins that bind carbohydrates, but many other compounds, including phytohormones (Delatorre et al., 2013) and nonprotein amino acids (Delatorre et al., 2007), have been observed to interact with lectins. Nevertheless, this is the first instance where the interaction between one lectin and a chromophore has been confirmed. No data are available about the biological role of pigment-proteins in marine sponges, but based on the chromophore-597/H-3 interaction, we speculate that H-3 may be involved in photoprotection of H. caerulea. The primary structure of H-3 reveals a unique amino acid sequence and shows no similarity with any family of animal lectins. However, H-3 showed high similarity with a putative protein encoded on the genome of the marine sponge A. queenslandica. Like H-3, pAqP it is a protein composed of two chains. The ␣-chain of pAqp has 197 amino acid residues and one signal peptide of 19 residues. Since H-3 N-terminal was blocked, it is likely that ␣H3 undergoes posttranslational processing similar to pAqP, being directed to extracellular matrix. Also, ␤pAqP is a polypeptide chain of 187 residues and one signal peptide of 18 amino acids, suggesting an addressing similar to ␤H-3. Curiously, both pAqP chains showed internal sequences not presented in H-3 chains. Both internal sequences flanking Nglycosylation sites in pAqP suggest a possible signal sequence. After glycosylation, we speculate that these internal sequences are removed by proteolytic cleavage because both sequences are

recognition sites to proprotein convertases, enzymes that catalyze maturation of proteins newly synthesized (Seidah and Chretien, 1997). Moreover, pAqP chains possess C-termini longer than the H-3 chain, suggesting another proteolytic processing at the Cterminal. In summary, we isolated and characterized a new GalNAcbinding blue lectin from the marine sponge H. caerulea. For the first time, the interaction between a lectin and a natural chromophore was demonstrated. H-3 does not show similarity to any known lectin, but it did present high identity to a putative protein from A. queenslandica. Together, H-3 and pAqP can be integrated into a new family of animal lectins. In our lab, we recently obtained crystals of H-3, and we are working to resolve its three-dimensional structure and consolidate the new animal lectin family. Acknowledgments This work was supported by the Brazilian agencies CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FUNCAP (Fundac¸ão Cearense de Apoio ao Desenvolvimento Científico e Tecnológico) and FINEP (Financiadora de Estudos e Projetos). The authors thank the National Museum–UFRJ, Rio de Janeiro, Brazil, for sponge identification. AHS, BSC, KSN are senior investigators of CNPq. References Ainouz IL, Sampaio AH, Freitas ALP, Benevides NMB, Mapurunga S. Comparative study on hemagglutinins from the red algae Bryothamnion seaforthii and Bryothamnion triquetrum. R Bras Fisiol Veg 1995;7(1):15–9. Betz SF. Disulfide bonds and the stability of globular proteins. Protein Sci 1993;2(10):1551–8. Bradford MM. A rapid and sensitive method for quatitation of micrograms quanties of proteins utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–534. Bornemann S, Rietschel B, Baltruschat S, Karas M, Meyer B. A novel polyacrylamide gel system for proteomic use offering controllable pore expasion by crosslinker cleavage. Electrophoresis 2010;31:585–92. Buck F, Luth CI, Bretting H. Comparative investigations on the amino acid sequences of different isolectins from the sponge Axinella polypoides. Biochim Biophys Acta 1991;1159:1–8. Buck F, Schulze C, Breloer M, Strupat K, Bretting H. Amino acid sequence of the D galactose binding lectin II from the sponge Axinella polypoides and identification of the carbohydrate binding site in lectin II and related lectin I. Comp Biochem Phys B 1998;121:153–60. Carneiro RF, Melo AA, Nascimento FEP, Simplicio CA, Nascimento KS, Rocha BAM, et al. Halilectin 1 (H-1) and Halilectin 2 (H-2): two new lectins isolated from the marine sponge Haliclona caerulea. J Mol Recognit 2013;26(1):51–8. Delatorre P, Rocha BAM, Souza EP, Oliveira TM, Bezerra GA, Moreno FBMB, et al. Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules. BMC Struct Biol 2007;7:52. Delatorre P, Silva-Filho JS, Rocha Bam, Santi-Gadelha T, Nóbrega RB, Gadelha CAA, et al. Interactions between indole-3-acetic acid (IAA) with a lectin from Canavalia maritima seeds reveal a new function for lectins in plant physiology. Biochimie 2013;95(9):1–7. Dodd RY, Maclenna AP, Hawkins DC. Haemagglutinins from marine sponges. Vox Sang 1968;15(5):386. Dresch RR, Zanetti GD, Lerner CB, Mothes B, Trindade VMT, Henriques AT, et al. ACLI, a lectin from the marine sponge Axinella corrugata: Isolation, characterization and chemotactic activity. Comp Biochem Phys C 2008;148:23–30. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28: 350–6. Freymann DM, Nakamura Y, Focia PJ, Sakai R, Swanson GT. Structure of a tetrameric galectin from Cinachyrella sp. (ball sponge). Acta Crystallogr D Biol Crystallogr 2012;68:1163–674. Glazer AN. Phycobilisomes: structure and dynamics. Annu Rev Microbiol 1982;36:173–98. Gouet P, Robert X, Courcelle E. ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 2003;31:3320–3. Gowda NM, Gowsami U, Khan MI. Purification and characterization of a T-antigen specific lectin from the coelomic fluid of marine invertebrate, sea cucumber (Holothuria scabra). Fish Shellfish Immunol 2008;24:450–8. Gundacker D, Leys SP, Schröder HC, Müller IM, Müller WEG. Isolation and cloning of a C-type lectin form the hexactinellid sponge Aphrocallistes vastus a putative aggregation factor. Glycobiology 2001;11:21–9.

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