Hemolytic activities in extracts of marine invertebrates from the Brazilian coast and isolation of two lectins from the marine sponge Cliona varians and the sea cucumber Holothuria grisea

Twenty species of marine invertebrates from the Brazilian coast were screened for hemagglutinating/ hemolytic activity. In at least twelve tested species, hemagglutinating activity was different for different blood types, suggesting the presence of lectins. Extracts from four species showed hemolytic activity. Two new lectins were purified from the marine sponge Cliona varians (CvL2) and sea cucumber Holothuria grisea (HGL). CvL-2 was able to agglutinate rabbit erythrocytes and was inhibited by galactosides. The hemagglutinating activity was optimal in pH neutral and temperatures below 70 °C. CvL-2 is a trimeric protein with subunits of 175 kDa. On the other hand, HGL showed both hemagglutinating and hemolytic activity in human and rabbit erythrocytes, but hemolysis could be inhibited by osmotic protection, and agglutination was inhibited by mucin. HGL was stable in pH values ranging from 4 to 10 and temperatures up to 90 °C. In electrophoresis and gel filtration, HGL was a monomeric protein with 15 kDa. CvL-2 and HGL showed different levels of toxicity to Artemia naplii. CvL-2 showed LC50 of 850.1 μg/mL, whereas HGL showed LC50 of 9.5 μg/mL.


INTRODUCTION
Marine biodiversity is the result of a long evolutionary history and extreme environmental conditions of pressure, light, oxygen, and temperature, giving rise to organisms with unique features (Ritchie et al. 2013).Marine life is much older than earthly life and has been evolving and diversifying longer (Burgess 2012).This amazing, but understudied, species diversity has provided a huge variety of molecules with biotechnological applications.Many of these compounds, which have already been isolated and studied, belong to marine invertebrates, such as sponges, cnidarians, mollusks, arthropods, echinoderms and tunicates (Rasjasa et al. 2011).Among these compounds are proteins (Pajic et al. 2002, Yoshida et al. 2007) and secondary metabolites (Kuramoto et al. 2004, Sipkema et al. 2005) with a wide range of biological activities.
Lectins are a group of carbohydratebinding proteins found in a wide range of organisms, including viruses, bacteria, fungi, plants, and animals.Lectins are involved in various biological functions, such as host defense, cell-cell interaction and folding of glycoproteins (Kilpatrick 2002, Sharon andLis 2004).Lectins from marine organisms are one of the promising candidates for useful therapeutic agents because carbohydrate structures, such as proteoglycans, glycoproteins, and glycolipids, have been implicated in certain cell types, and their physiological and pathological functions include host-pathogen interactions and cell-cell communication (Ogawa et al. 2011).
In particular, marine invertebrate lectins have attracted great interest due to their biotechnological properties.For instance, a lectin isolated from the sea cucumber Cucumaria echinata (CEL-III) was expressed in transgenic mosquitoes and impairs the development of the Malaria parasite (Yoshida et al. 2007).Lectins isolated from the marine sponges Haliclona cratera and Craniella australiensis displayed cytotoxic effect against malignant cells and mitogenic response towards BALB/c splenocytes, respectively (Pajic et al. 2002, Xiong et al. 2006).
The growing applications of lectins underscore the potential economic importance of the discovery of novel species for biotechnology.To date, the tropical sponge Cliona varians possesses one isolated lectin, CvL-1.CvL-1 is a galactose-binding lectin with cytotoxic effect on pathogenic bacteria, such as Bacillus subtilis and Staphylococcus aureus (Moura et al. 2006).Furthermore, the sea cucumber Holothurea grisea possesses two isolated lectins: HGA, an anti-inflammatory lectin of 228 kDa, and HGA-2, a C-type lectin of 34 kDa (Moura et al. 2012, Melo et al. 2014).In the present work, we screened twenty Brazilian species of marine invertebrates for their hemagglutinating/hemolytic activity, and found two new lectins isolated from C. varians and H. grisea.
The decapods Callinectes danae were collected at Barra do Ceará Beach, Fortaleza, state of Ceará, Brazil.SCREENING OF HEMAGGLUTINATING/HEMOLYTIC ACTIVITIES Specimens of the marine sponge Cliona varians and the sea cucumber Holothurea grisea were collected at Santa Rita Beach, state of Rio Grande do Norte, Brazil.
The animals were transported in a thermal box at 4 °C and then stored at -20 °C until use.All collections were performed on sunny days, when water temperature was between 25 °C and 27 °C.

PREPARATION OF AQUEOUS EXTRACTS
Marine sponges were triturated in grail and homogenized in Tris buffer 50 mM, pH 7.6, containing NaCl 150 mM and CaCl 2 20 mM (TBS/ Ca 2+ ) at a ratio of 1:2 (w/v).The mixtures were filtered and centrifuged at 8000 xg for 20 min at 4 °C.Supernatants were stored at -20 °C until use.
The cnidarians were freeze-dried and triturated until a fine powder was obtained.The powders were homogenized in TBS/Ca 2+ at a ratio of 1:2 (w/v).The mixtures were filtered and centrifuged at 8000 xg for 20 min at 4 °C.The supernatants were stored at -20 °C until use.
The mollusks and echinoderms Holothurea grisea and H. arenicola were cut into small pieces, triturated in mortar and pestle, homogenized in TBS/Ca 2+ and filtered.After centrifugation, supernatants were stored at -20 °C until use.
Specimens of Echinometer lucunter were maintained on ice until dead for inanition.Then, peristomial membrane was cut around the mouth and the Aristotle's lantern was removed, and the celomatic plasma was collected by pipetting.The coelomic plasma was allowed to clot for 1h at 0 °C.The supernatants were stored at -20 °C until use.
The swimming crabs C. danae averaging 5 cm in length were maintained in ice.The hemolymph was collected from the cheliped using a sterile syringe, and allowed to clot for 1h at 0 °C.The supernatants were stored at -20 °C until use.
Crude extracts were used within three months after preparation.

HEMAGGLUTINATING/HEMOLYTIC AND INHIBITION ASSAYS
The hemagglutinating/hemolytic activities were assayed in microtiter V plates (Nunc, Denmark) as described by Sampaio et al. (1998).Native blood cells (Human ABO and rabbit erythrocytes) were treated with papain and trypsin.One hemagglutinating unit (HU) was defined as the reciprocal of the highest dilution giving positive hemagglutination.Hemolytic activity was examined by visual examination of lysis of the erythrocytes under conditions similar to those of the hemagglutination assay.
In the assay using the lectin purified from Holothuria griea, erythrocytes were osmotically protected.Hemagglutinating activity was evaluated in the presence of dextrose 5.5%.
PURIFICATION OF HOTOTHURIA GRISEA HEMOLYTIC LECTIN (HGL) Specimens of H. grisea were cut into small pieces.The pieces were washed in distilled water and freeze-dried.Pieces were then triturated in mortar and pestle and extracted at a ratio of 1:2 (w/v) with 0.05 M Tris-HCl buffer pH 7.6 containing NaCl 0.15 M (TBS) for 2h at room temperature.After centrifugation for 30 min at 8000 xg at 4 °C, the supernatant (crude extract) was partitioned with n-butanol 50% and ethanol 75% and then precipitated with acetone at 2.0 vol.The supernatant (F2) was dried in a vacuum concentrator and solubilized in a small volume of TBS.After centrifugation, F2 was RANIERE M. MOURA et al.
applied to a TSK gel G3000SWXL (1.28 × 30 cm) column coupled to an ÄKTA purifier system.The column was equilibrated and eluted with TBS at a flow rate of 1 mL/min.Chromatography was monitored at 280 nm, and 1 mL fractions were collected.
PURIFICATION OF CLIONA VARIANS LECTINS (CVL-1 AND CVL-2) C. varians crude extract was prepared as described above.The crude extract was precipitated with acetone at 1.0 vol.After centrifugation, the pellet was recovered with 0.05 M Tris buffer, pH 7.0, containing 1M (NH 4 ) 2 SO 4 (Tris/(NH 4 ) 2 SO 4 ).This CvL--enriched fraction showed a higher level of hemagglutinating activity to rabbit trypsin-treated erythrocytes.CvL--enriched fraction was then applied on Phenyl-Sepharose 6B column (1.0 x 6.0 cm) previously equilibrated with Tris/(NH 4 ) 2 SO 4 .The column was washed with the same buffer, and retained proteins were eluted with linear gradient of (NH 4 ) 2 SO 4 (1 -0M) in 0.05 M Tris, pH 7.0, at a flow rate of 2 mL/min.Fractions of 2 mL were manually collected.Active fractions were pooled, dialyzed against deionized water, and freeze-dried.
Freeze-dried proteins were solubilized with TBS/Ca 2+ at a concentration of 5 mg/mL.After centrifugation, 1 mL was injected into a Sephacryl S-300 HR 16/60 column (1.6 x 60.0 cm) coupled to an ÄKTA purifier system.The column was equilibrated and eluted with TBS at a flow rate of 1 mL/min.Chromatography was monitored at 280 nm, and 3-mL fractions were collected.

EFFECTS OF PH, TEMPERATURE AND CATIONS ON HEMAGGLUTINATING/HEMOLYTIC ACTIVITY OF CVL-2 AND HGL
The effect of pH, temperature and divalent ions on hemagglutinating/hemolytic activity was evaluated following Sampaio et al. (1998).

MOLECULAR MASS ESTIMATION OF HGL AND CVL-2
Sodium dodecyl sulfate polyacrylamide (10% and 15%) gel electrophoresis (SDS-PAGE), in the absence and presence of β-mercaptoethanol, was conducted as described by Laemmli (1970).The molecular mass of lectin and its subunits was estimated by comparing mobility of the bands with the following protein markers (kDa): Bovine serum albumin (Mr 67), Ovalbumin (Mr 43), Carbonic anhydrase (Mr 29) Ribonuclease (Mr 13.7) and Aprotinin (Mr 6.5).Proteins were detected by staining with 0.1% Coomassie brilliant blue R-250.

LETHALITY TEST IN ARTEMIA
Lethality test on Artemia nauplii was performed in accordance to pre-established methods (Carneiro et al. 2013).
Aqueous extracts of C. varians and H. grisea were dialyzed and freeze-dried.Extracts were prepared by homogenization with artificial sea water (ASW) at a concentration of 5 mg/mL.The purified lectins were solubilized in ASW at 1 mg/mL.
The tests were done on board 24-well Limbro plates such that each well contained 2 mL of artificial sea water (ASM) with 10 nauplii.Specific volumes of samples were added to each well to SCREENING OF HEMAGGLUTINATING/HEMOLYTIC ACTIVITIES obtain concentrations of 10, 25, 50 and 100 µg/mL for each sample.The experiments were performed in triplicate with control containing only 2 mL of ASW and 10 nauplii.Dead nauplii were counted after 24h.From these data, we calculated the percentage of deaths for each concentration and LC 50 values were determined by probit analysis, as described by Finney (1971).

LECTIN AND HEMOLYTIC ACTIVITY OF MARINE INVERTEBRATE AQUEOUS EXTRACTS
Twenty species of marine invertebrates were screened for their hemagglutinating/hemolytic activity, and the results are shown in Table I.
Eight of the nine species of sponges tested, exhibited hemagglutinating activity, and all species showed preference for some group/treatment of blood, suggesting the presence of lectin like molecules.
Species of the genus Holothuria showed both hemagglutinating and hemolytic activities.After agglutination, rapid hemolysis was observed.However, hemolysis was able to be halted by crude extract precipitation with 1 vol of acetone.In the extracts of the marine sponge Amphimedon viridis and the mollusk Ischinachiton pectinatus, hemolysis was also observed, but it could not be stopped.
Among the twenty species tested, we selected the two most abundant species, C. varians and H. grisea, for lectin purification and toxicity assay.The crude extract of H. grisea showed a higher level of hemagglutinating/hemolytic activity to all tested erythrocytes.After extract partition with n-butanol and ethanol, hemagglutinating/hemolytic activity was concentrated in Fraction 2 (F2).Thus, F2 was submitted to size-exclusion chromatography on TSKgel, where peak 1 corresponded to the pure hemolytic lectin HGL (Fig. 1).This process resulted in purifi cation of 35% with a 53% yield (Table II).Approximately 100 mg of CvL-enriched fraction were applied to the column (1.0 × 6.0 cm) equilibrated with Tris/(NH 4 ) 2 SO 4 .The lectin was eluted with linear gradient of (NH 4 ) 2 SO 4 (1 -0M) in 0.05 M Tris, pH 7.0, and fractions (2.0 mL each) were monitored at 280 nm.The hemagglutinating activity was detected in the presence of 3% rabbit trypsin-treated erythrocytes (B) Size exclusion chromatography on Sephacryl S300 profi le.Approximately 5 mg of proteins retained on Phenyl-Sepharose were applied to the column equilibrated with TBS/ Ca 2+ , pH 7.6, and fractions (3.0 mL each) were monitored at 280 nm.The hemagglutinating activity was detected in the presence of 3% trypsin-treated rabbit erythrocytes.SCREENING OF HEMAGGLUTINATING/HEMOLYTIC ACTIVITIES of acetone precipitation, hydrophobic interaction chromatography (Fig. 2A) and size-exclusion chromatography (Fig. 2B).Compared to the activity in crude extract, the specific lectin activity was increased 21 times by purification procedure.Purified CvL-2 represented 4.5 % of the total HA of the extract (Table III).This procedure also resulted in the copurification of another lectin from C. varians: CvL-1 (Fig. 2B).CvL-1 was purified 60 times from the crude extract and represented 48 % of total HA of the extract (Table III).

MOLECULAR MASS ESTIMATION OF THE PURIFIED LECTINS
The apparent molecular mass of the purified native HGL was estimated by gel filtration on calibrated TSKgel G3000SWXL column.It was calculated as 17 kDa (data not shown).In addition, on SDS-PAGE, under reducing and nonreducing conditions, HGL showed an apparent molecular mass of 15 kDa (Fig. 3A).These results suggest that HGL is a monomeric lectin, devoid of intermolecular disulfide bonds.On the other hand, native CvL-2 is a 465 kDA protein by native PAGE (Fig. 3B).On   size-exclusion, CvL-2 was eluted in void-volume, suggesting a molecular mass higher than 1,000 kDa (data not shown).SDS PAGE of purified lectin showed a single band of 175 kDa, under reducing and nonreducing conditions (Fig. 3C).These data suggest that CvL-2 presents anomalous behavior.While appears as a trimeric protein on N-PAGE, it shows multimeric form on size-exclusion chromatography.RANIERE M. MOURA et al.

LECTINS
HGL quickly hemolyzed all tested erythrocytes.However, at the beginning of the hemolysis process, it was possible to observe the agglutination of erythrocytes.We were un successful in achieving the inhibition of hemolytic activity with sugars, therefore, we conducted an osmotic protection assay.With this procedure, it was possible to observe that the dextrose was able to protect against erythrocyte lysis at the last dilution of HGL.This allowed us to visualize the agglutination by optical microscopy (Fig. 4) and perform the inhibition test.Hemagglutinating activity of HGL was inhibited by PSM and fetuin with minimal inhibitory concentration (MIC) at 0.31 mg/mL and 2.5 mg/ mL, respectively (Table IV).CvL-2 strongly agglutinates trypsin-treated rabbit erythrocytes and, to a lesser degree, trypsin-treated human erythrocytes derived from blood type A. Hemagglutinating activity of CvL-2 was inhibited by galactosides and porcine stomach mucin (PSM).GalNAc and PSM were potent inhibitors of CvL-2 activity with MIC at 0.2 mM and 9.7 µg/mL, respectively.Galactose (MIC=12.5 mM), α-methyl-D-galactopyranoside (MIC=6.25 mM) and lactose (MIC=3.1 mM) were all able to inhibit CvL-2 (Table IV).
EFFECTS OF PH, TEMPERATURE AND DIVALENT CATIONS Hemolytic activity of HGL was not inhibited when previously treated with EDTA.Although we added the divalent cations Ca 2+ , Mg 2+ and Mn 2+ , no change in protein activity was observed.HGL hemolytic activity was stable in all ranges of tested pH (4-10).HGL exhibited high thermostability.The hemolytic activity was unaffected after exposure of the lectin at 90 °C, and after 1h at 100 °C, the activity decreased to 50%.
Hemagglutinating activity of CvL-2 was completely abolished after dialysis against EDTA.The activity of the lectin was restored when EDTA was removed and 5 mM CaCl 2 was added to the protein solution.The lectin activity of CvL-2 was optimum on neutral pH (7).The activity rapidly decreased when the lectin was incubated in basic or acidic pH.CvL-2 showed relative thermostability.Its activity was stable at 50 °C, but gradually decreased with increasing temperature.At 90 °C, the activity remained at 25%, but it was completely abolished at 100 °C.SCREENING OF HEMAGGLUTINATING/HEMOLYTIC ACTIVITIES TOXIC EFFECTS OF CRUDE EXTRACTS AND LECTINS PURIFIED FROM C. VARIANS AND H. GRISEA Extracts from C. varians and H. grisea showed different levels of toxicity on Artemia nauplii.Crude extract of H. grisea was very toxic (LC 50 = 8.1±3.0 μg/mL), whereas crude extract of C. varians showed low toxicity (LC 50 = 116±23.3μg/mL).The toxic effects were observed to be dose-dependent.The lectins HGL and CvL-1 showed potent activity against Artemia nauplii.LC 50 values obtained were 9.5 µg/mL and 5.6 µg/mL, respectively.However, CvL-2 showed no toxicity against Artemia nauplii (LC 50 = 850.1±3.4 μg/mL).

DISCUSSION
In at least twelve tested species, hemagglutinating activity was different for different blood types, suggesting the presence of lectin-like molecules in these species; namely, hemagglutinating activity was observed in 60% of the tested species.This value is similar to those found in other screenings of hemagglutinating/hemolytic activity of marine invertebrates (Dresch et al. 2005, Mojica et al. 2005).Furthermore, while hemolytic molecules, for the most part remain unknown, hemolytic activity was found in the extracts of some studied species, and HGL; which seems to be a hemolytic agent, was isolated in Holothurea grisea.
Toxic activity is a common factor among lectins (Pajic et al. 2002, Queiroz et al. 2008, Santos et al. 2010).Similar to lectins isolated from Haliclona caerulea (Carneiro et al. 2013), Axinella corrugata (Dresch et al. 2011) and Aplysia kurodai (Kawsar et al. 2010), HGL showed a high level of cytotoxicity against Artemia nauplii.However, hemolysis caused by lectin is rare, and few lectins have shown hemolytic activity (Hatakeyama et al. 1994, Sudhakar andVincent 2014).The lectin isolated from the sea cucumber Cucumaria echinata (CEL-III) showed both hemolytic and toxic activities (Hatakeyama et al. 1994, Oda et al. 1997).CEL-III is an R-type lectin that is Ca 2+ -dependent and gal/galNAc-binding (Nakano et al. 1999).These activities are mediated by binding of the protein to the specific carbohydrate chain on the target cells, followed by the formation of ion-permeable pores in the cell membrane through protein oligomerization (Hatakeyma et al. 1995).We were not able to determine the mechanism which mediates HGLinitiated hemolysis.However, both the agglutination of erythrocytes after osmotic protection and the HGL inhibition by PSM, indicated that the binding of HGL to carbohydrates in the erythrocyte membranes is the first step in hemolytic activity.
HGL showed singular stability, maintaining its activity in a broad range of pH and temperature.Usually, lectins from marine invertebrates possess optimal activity on basic-neutral pH and thermal stability up to temperatures of 70 °C.
Lectins isolated from sea cucumbers are generally ion-dependent proteins, many of which sharing structural factors that allow them to be grouped into the C-type super family (Himeshima et al. 1994, Hatakeyama et al. 2002), but HGL could not be grouped into the C-type super family .In general, C-type lectins bind to monosaccharides, such as mannose and galactose.HGL is not an iondependent lectin and does not bind to galactose or mannose.On the other hand, CvL-2 could be inhibited by EDTA, indicating that CvL-2 activity was Ca 2+ -dependent.Lectins isolated from the sponges Aplysina lawnosa, Aplysina archeri and Aphrocallistes vastus also presented Ca 2+dependent activity (Miarrons andFresno 2000, Gundacker et al. 2001).However, it is impossible to define CvL-2 as a C-type lectin without knowledge of its primary structure.
CvL-2 was inhibited by lactose at MIC=3.1 mM, whereas CvL-1 showed MIC=25 mM to some disaccharides (Moura et al. 2006).CvL-1 showed a slight affinity to glucose and its derivatives (Moura et al. 2006), but CvL-2 did not.The inhibition test for both lectins suggests that C-4 hydroxyl represents a point of differential recognition for CvL-1 and CvL-2.For CvL-2, C-4 hydroxyl likely interacts directly with CRD, participating in recognition, since galactose, but not glucose, was able to inhibit the activity of CvL-2.GalNAc was the powerful inhibitor for CvL-2 activity, most likely because the presence of N-Acetyl group increases the number of interactions between CvL-2 CRD and sugar.In fact, several lectins from marine invertebrates have presented affinity to GalNAc (Belogortseva et al. 1998, Beisel et al. 1999).
Furthermore, similar to other lectins from marine invertebrates (Moura et al. 2006, Dresch et al. 2008), optimal activity of CvL-2 was observed in neutral pH, probably because that pH represents the natural environment of sponges.
Many extracts of aquatic organisms have been tested against Artemia nauplii and showed toxic activities (Thompson et al. 1985, Carbalho et al. 2002).Some of these compounds have already been isolated and evaluated in other models.The results have shown a range of biological activities, such as anticancer, suppression of the HIV virus, anti-inflammatory and proinflammatory activity (Herencia et al. 1998, McCune et al. 1989).In a previous study, CvL-1 was able to inhibit the growth of human leukemia cells, triggering the signaling of programmed cell death (Queiroz et al. 2008).CvL-1 also showed a high level of toxicity against Artemia nauplii.The new lectins isolated in this work showed different levels of toxicity to Artemia nauplii.HGL was highly toxic and could, like CvL-1, be a powerful biotechnology tool in cancer research.On the other hand, CvL-2 showed low toxicity effects and is expected to have no effect on malignant cells and inflammatory response.
In summary, hemagglutinating activity was observed in about 60% of tested species, indicating that marine invertebrates from the Brazilian coast are a rich and untapped source of new lectins.Furthermore, two new lectins were isolated, and one, HGL, is a rare lectin with hemolytic activity.Thus, our cataloging of lectin activity on marine invertebrates leads us to novel features associated with these marine organisms from a biotechnological perspective.SCREENING OF HEMAGGLUTINATING/HEMOLYTIC ACTIVITIES proteína trímero com subunidades de 175 kDa.Por outro lado, HGL apresentou atividade hemaglutinante e hemolítica em eritrócitos humanos e de coelho, mas a hemólise pôde ser inibida por proteção osmótica, enquanto que a aglutinação foi inibida por mucina.

Figure 1 -
Figure 1 -Purifi cation of HGL.TSKgel profi le of F2 from H.grisea. Approximately 0.5 mg of F2 was applied to the column previously equilibrated with TBS pH 7.6, and fractions (1.0 mL each) were monitored at 280 nm.The hemolytic activity was detected in the presence of 3% rabbit erythrocytes.

Figure 2 -
Figure 2 -Purifi cation of CvL-2.(A) Phenyl Sepharose 6B chromatography profi le.Approximately 100 mg of CvL-enriched fraction were applied to the column (1.0 × 6.0 cm) equilibrated with Tris/(NH 4 ) 2 SO 4 .The lectin was eluted with linear gradient of (NH 4 ) 2 SO 4 (1 -0M) in 0.05 M Tris, pH 7.0, and fractions (2.0 mL each) were monitored at 280 nm.The hemagglutinating activity was detected in the presence of 3% rabbit trypsin-treated erythrocytes (B) Size exclusion chromatography on Sephacryl S300 profi le.Approximately 5 mg of proteins retained on Phenyl-Sepharose were applied to the column equilibrated with TBS/ Ca 2+ , pH 7.6, and fractions (3.0 mL each) were monitored at 280 nm.The hemagglutinating activity was detected in the presence of 3% trypsin-treated rabbit erythrocytes.

Figure 4 -
Figure 4 -Hemagglutinating activity of HGL under osmotic protection with 5.5% dextrose.A) Hemagglutinating of rabbit erythrocytes treated with trypsin in the presence of HGL; B) Control group: erythrocytes in saline.

TABLE I Screening of aqueous extracts of 20 marine invertebrate species from the Brazilian coast for hemagglutinating/ hemolytic activity. Hemagglutinating activity is described in titer
. N-native erythrocytes; T-trypsin-treated erythrocytes; P-papain-treated erythrocytes; L-hemolytic activity observed.RANIERE M. MOURA et al.PURIFICATION OF HEMOLYTIC LECTIN FROM HOLOTHURIA GRISEA