ISJ 16: 15-24, 2019 ISSN 1824-307X RESEARCH REPORT Sabella spallanzanii mucus contain a galactose-binding lectin able to agglutinate bacteria. Purification and characterization M Cammarata 1 * ,# , G Benenati 2,# , M Dara 1 , MG Parisi 1 , D Piazzese 1 , F Falco 3 , L Stabili 4,5 # Equal contribution 1 Department of Heart and Marine Science DISTEM, Marine Immunobiology Laboratory, University of Palermo, , Italy 2 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies STEBICEF, University of Palermo, Italy 3 Istituto per le Risorse Biologiche e le Biotecnologie Marine (IRBM), Units of Capo Granitola Italy 4 Istituto per l’Ambiente Marino Costiero, U.O.S. di Taranto, CNR, Taranto, Italy 5 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy Accepted March 04, 2019 Abstract Lectins are present in almost all living organisms and are involved in several biological processes, including immune responses. In the present study, a calcium dependent galactose-binding lectin exhibiting an apparent MW of 43 kDa has been characterized and purified from the mucus of the polychaete Sabella spallanzanii by using both affinity chromatography and high-pressure liquid chromatographic methods. Its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or EDTA. The activity was optimal at temperature values comprised between 4 and 18 °C, maintain a 50% of activity between 20 and 37 °C, was significant deleted after exposure at 50 °C, and was depleted at 90 °C. The S. spallanzanii Galactose-Binding Lectin (SsGBL) was able to agglutinate bacteria and to preferentially recognize Gram-negative bacteria. The strongest agglutinating activity was observed towards Vibrio alginolyticus and Escherichia coli, by contrast mucus agglutinated in a lesser extent both Aeromonas hydrophyla and the Gram- positive Micrococcus lysodeikticus thus suggesting its involvement in host pathogen interactions. Key Words: mucus; hemagglutinin; bacteria; galactose-binding lectin; S. spallanzanii Introduction Lectins are multifamily proteins present in almost all living organisms and due to their carbohydrate binding ability are involved in several biological processes (Kaltner and Stieltorfer, 1998; Kilpatrick, 2002), including development, cell adhesion, glycoproteins interactions (Kaltner and Stierstorfer, 1998; Kilpatrick, 2002), and immune responses (Liao et al., 1994; Arason, 1996). Lectins and sugars constitute an evolutionary conserved recognition system, involved in innate immunity, able to mediate several effector functions. These ___________________________________________________________________________ Corresponding author: Matteo Cammarata Department of Heart and Marine Science DISTEM Marine Immunobiology Laboratory University of Palermo Viale delle Scienze Ed. 16, Palermo, Italy E-mail: matteo.cammarata@unipa.it activities include agglutination, immobilization and opsonization towards microbial pathogens and complement activation, by either recognition of glycans exposed on potential pathogens either immunoregulation binding to carbohydrates on immunocompetent cells surfaces (Turner, 1996; Kilpatrick, 2002; Loris, 2002; Fujita et al., 2004; Sharon and Lis, 2004; Vasta et al., 2004). Lectins have been classified into various structural families such as C-type lectin, galectin and R-type lectin. The lectins are distinguished on the basis of conserved amino acid sequence motifs in their carbohydrate recognition domain (CRD), structural folds and calcium requirements (Aranson, 1996; Turner, 1996; Fujita et al., 2004; Sharon and Lis, 2004; Vasta et al., 2004). Galactose-binding lectins have been documented both in vertebrates and in invertebrates, and their involvement in humoral 15 immunological processes is well described (Arason et al., 1996; Vasta et al., 2004). Moreover, many of them are able to bind β-galactoside carbohydrates other than monosaccharide galactose (Hirabayashi et al., 2002; Vasta et al., 2004). Much evidence exists about the presence of galactose-binding lectins in marine invertebrates. In phylum Annelida, a 29 kDa galactose-binding lectin was characterized in the earthworm Lumbricus terrestris (class Oligochaeta) (Hirabayashi et al., 1998). The primary structure of the earthworm lectin belongs to R-type lectin family that is involving ricin B-chain. On the other hand, annelid lectins that recognize galactose and others sugars were isolated from various marine worms including polychaeta and oligochaeta. Each lectin has different characteristics on carbohydrate-binding specificities, metal requirement and primary structure. Isolation, physicochemical properties, and, in some cases, biological activity and primary structure of such lectins have been described. Amphitritin, a Ca 2+ - independent N-acetyl D-galactosamine-binding lectin with molecular mass of 30 kDa was the first hemagglutinin isolated from a sea worm Amphitrite ornata (Garte and Rissel, 1976). A 30 kDa β- galactose-specific lectin was isolated and characterized from the sea worm Chaetopterus variopedatus (Mikheyskaya et al., 1995). Curiously, this lectin revealed cytopathic effect induced by human immunodeficiency virus (Wang et al., 2006). D-galactose-binding lectins (33-35 kDa) were isolated from body walls of echiuroid (Urechis unicinctus; oligochaeta) and marine worms (Neanthes japonica and Marphysa sanguinea) (Ozeki et al., 1997). Another 32 kDa D-galactose- binding lectin isolated from the marine worm Perinereis nuntia was shown to have QxW sequence in the polypeptides (Kawsar et al., 2009). This sequence motif was seen in R-type lectin family. Marine duster worm, Sabella spallanzanii (phylum Annelida, family Sabellidae) is a representative tube worm in the Mediterranean bay. Its glandular epithelium secreting mucus often appears conspicuous and forming the so called “ventral shield”. Mucus production, as in many invertebrates, constitutes a key factor determining the ability of many polychaete species to survive in their environment (Beckwith, 1999; Smith, 2002; Davies and Ogawa, 2011). As reported by Storch (1988) mucus intervenes in fertilization and egg protection, consolidates the tunnel wall of burrowing polychaetes and may also play a role in the absorption of metabolites (Mouneyrac et al., 2003; Mastrodonato et al., 2005, Dales, 1961; Stabili et al., 2009). Their defensive functions, such as cytotoxicity and lysozyme-like activity, (Canicattì et al., 1992; Stabili et al., 2009; Giangrande et al., 2014) inhibit in vitro the growth of Vibrio anguillarum, Vibrio harveyi, Pseudomonas aeruginosa and Candida albicans (Stabili et al., 2011). In the present study, we further investigated the defensive role of S. spallanzanii mucus and we reported the identification, purification and characterization of a novel galactose-binding lectin with agglutinating activity against rabbit red blood cells and several bacteria. This lectin was isolated by both affinity chromatography and high-pressure liquid chromatographic methods. Results are discussed in the light of elucidating the involvement of mucus in prevention of pathogenic microorganism proliferation. Material and Methods Chemicals, molecular biology reagents Unless otherwise specified, chemicals and reagents were from Sigma-Aldrich (USA). Animals, mucus collection and preparation Sampling was undertaken in the harbor of Brindisi (Southern Adriatic Sea, Italy) using SCUBA equipment (depth range = 5-15 m). About 200 adult specimens of Sabella spallanzanii were collected and transferred to the laboratory. In order to stimulate the secretion of the mucus, all the individuals have been removed from the tube where they lived and kept for 30 min in a Petri dish. Within the secreted mucus, we checked for trapped material by microscopical observations, whilst we excluded any contamination of other excretion products by pH measurements. Secreted mucus was collected and centrifuged at 12000 xg for 30 min at 4 °C and stored at -80 °C until used. It was ten folds diluted in tris-buffered saline (TBS) and filtered through 0.2 µm pore size before performing affinity chromatography. Hemagglutination assay Rabbit and sheep red blood cells (RaRBC and SRBC, supplied by Istituto Zooprofilattico della Sicilia) were washed three times in phosphate buffered saline (PBS), centrifuged at 500 xg for 10 min at 4 °C and suspended at 1% in PBS containing 0.1% (w/v) gelatin. A volume (25 μl) of S. spallanzanii mucus or 25 μl of the dialyzed purified S. spallanzanii Galactose-Binding Lectin (SsGBL) were serially (2-fold) diluted in PBS-gelatin in 96- well round-bottom microtiter plates (Denmark), and an equal volume of erythrocytes suspension was added. The hemagglutinating titer (HT) was measured after 1 hour incubation at 37 °C and expressed as the reciprocal of the highest dilution showing clear agglutination (Ballarin et al., 2008). Physical and chemical characterization To examine divalent cation requirement for mucus hemagglutination activity (HA), CaCl2 and MgCl2 were added to the assay medium to obtain 3 mM each one final concentration. EDTA (10 mM) or EGTA (10 mM) were used to examine the effect of Ca 2+ versus Mg 2+ depletion. To examine the thermolability, mucus samples were incubated at 4, 10, 18, 37, 50, 70, and 90 °C for 20 min and cooled down for 10 min on ice before testing the HA. Carbohydrate specificity Hemagglutinating activity was assayed against RaRBC in the presence of serially diluted saccharides as potential inhibitors (Ballarin et al., 2008). A volume (25 μl) of S. spallanzanii mucus or 25 μl of the purified S. spallanzanii Galactose- Binding Lectin (SsGBL) and 25 μl of a serially 16 diluted sugar. Finally, an equal volume of erythrocytes suspension was added and after 1 hour incubation at 37 °C the HT was evaluated. Inhibition experiments were carried out using decreasing concentrations (starting from 130 mM in PBS pH 7.4, 3 mM CaCl2, 1% gelatin) of monosaccharides (L-fucose, L-rhamnose, D- galactose, D-glucose, D-mannose, N- Acetylglucosamine) and disaccharides (Lactose, and Lactulose). The same procedure was performed in the inhibition experiments having bacteria as target. Bacterial suspensions and agglutination In order to evaluate the hemagglutinating activity the following bacterial strains were employed: Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophyla, Staphylococcus aureus and Micrococcus lysodeikticus. Bacteria were grown to log phase in tryptic soy broth (TSB) containing 3% NaCl at 25 °C, with continuous shaking (120 rpm) in a Gallenkamp incubator. Log phase was estimated by absorbance at 600 nm. The correspondence between cell number and spectrophotometric absorbance have been determined by serial dilution plate count method. Bacteria were killed with heat incubating them at 121 °C, for 20 min, at 1 atm. For the agglutination assay, they were washed three times in sterile PBS, suspended in PBS containing 0.1% (w/v) gelatin to obtain 1x10 7 bacteria/ml and dispensed in 96 wells plate. Plates were incubated at 18 °C over night. Lectin purification Lectin was isolated by a two-steps chromatography procedure. The first consisted of a galactose-agarose affinity chromatography column with elution with 0.1 M galactose in TBS, 3 mM CaCl2, as previously reported (Salerno et al., 2009). The elution step was monitored by absorbance at 280 nm and protein concentration in collected fractions was evaluated through Bradford method (1976). After dialysis in TBS, 3 mM CaCl2, these were tested for hemagglutinating activity towards rabbit erythrocytes, and those that exhibited the highest activity were pooled and analysed by SDS- PAGE (Laemmli, 1970). In the second step, the collected fractions from the chromatographic procedure exerting hemagglutinating activity were applied to a High- Pressure Liquid Chromatography Size Exclusion Column BioSuite 250–10 µm SEC 7.5 x 300 mm Waters, 350 psi pressure, 280/254 nm (mAU) and analysed by HPLC method. Phosphorylase b (97kDa), bovine serum albumin (BSA, 67kDa), enolase (46.7kDa), myoglobin A (18.7kDa) and RNaseA (13.7kDa) were used as calibration standards. Affinity column purified fractions were then applied to High Pressure Liquid Chromatography Size Exclusion Column BioSuite 250–10 µm SEC 7.5 x 300 mm Waters, 350 psi pressure, 280/254 nm (mAU). Phosphorylase b (97kDa), bovine serum albumin (BSA, 67kDa), enolase (46.7kDa), myoglobin A (18.7kDa) and RNase A (13.7kDa) were used as calibration standards (Fig. 3B). Table 1 Range of hemagglutinating activity (titer 1 ) of S. spallanzani mucus and the purified lectin (25 µg/ml) towards various erythrocytes and bacteria Erythrocytes Mucus Isolated fraction Rabbit Red Blood Cells 512-1024 32-128 Sheep Red Blood Cells 0-2 - Escherichia coli 128- 256 16-32 Vibrio alginolyticus 128-512 16-32 Aeromonas hydrophila 64-128 8-16 Staphilococcus aureus NA NA Micrococcus lysodeikticus 32-64 8-16 Protein content estimation Protein content was estimated according to the Bradford method using BSA as a standard. Undiluted mucus showed a protein content of about 0.6 mg/ml while the best eluted chromatographic fraction had a protein concentration of about 0.2 mg/ml. Polyacrylamide gel electrophoresis SDS-PAGE (16%) was carried under reducing (5% mercaptoethanol) and non-reducing conditions. To evaluate the molecular size, gels were calibrated with low molecular weight (6.5-66 kDa) standard proteins. Proteins were stained with Coomassie brillant Blue R250. Results Mucus hemagglutinating activity Mucus agglutinating activity was tested towards both erythrocytes and bacteria. It showed almost no activity when sheep erythrocytes were used as target cells (HA titer = 2). Otherwise, this matrix had a strong agglutinating activity when rabbit red blood cells were used in the test showing an average agglutination titer of 512 (Table 1). This activity was calcium dependent because it was strongly affected by calcium depletion when 10 mM EDTA or 10 Mm EGTA and 3 mM magnesium were added to the hemagglutination assay medium. RaRBC hemagglutinating activity thermolability was tested performing the assay after 20 min pre- incubation of mucus at different temperatures. The optimum of the activity was recorded when temperature ranged between 4 and 18 °C and decreased after 20 min pre-incubation temperature ranging from 37 to 90 °C (Fig. 1). Almost no loss of biological activity was detected after two months storage of samples at -80 °C. 17 Fig. 1 S. spallanzanii mucus preincubated 20 min at different temperature was tested towards RaRBC. It to have an optimum agglutination temperature ranging between 10 °C and 18 °C and a reduced but not completely deleted activity even after 20 min pre-incubation step from 37 °C up to 90 °C Mucus was able to preferentially recognize Gram-negative bacteria; indeed, the strongest agglutinating activity was observed towards V. alginolyticus and E. coli, by contrast mucus agglutinated in a lesser extent A. hydrophyla and the Gram-positive M. lysodeikticus and did not agglutinate the Gram-positive S. aureus (Table 1). Carbohydrates inhibition test was performed by adding several carbohydrates in decreasing concentrations (final concentration ranging from 130 to 4 mM) to the assay medium. Galactose and at lesser extent fucose revealed to have inhibition activity even at the lowest concentration (8 mM Galactose, 16 mM Fucose) used in the hemagglutination assay (Table 2). SsGBL purification and characterization The SsGBL has been purified starting from a 20 ml diluted collected mucus applied on galactose- agarose column. The profile of the affinity purification is shown in Figure 2. In a typical isolation, the eluted fractions, having a protein concentration ranging from 0.08 to 0.18 mg/ml, represented approximately 10-30% of the total mucus protein content loaded onto the column (0.61 mg/ml). The recovery in terms of hemagglutinating activity was about 25% and the 3% after HPLC elution (Table 3). The galactose eluted fractions, having the highest protein concentration, showed similar average hemagglutinating activity towards RaRBC that ranged from 32 to 64 (Table 2). The action of the active fractions was calcium dependent because it appeared magnified when the medium contained 3 mM calcium and was heavily affected by the addition of 10 mM EDTA or 10 mM EGTA, 3 mM magnesium, as already observed for mucus extracts. Electrophoresis analysis on SDS-PAGE revealed that the purified lectin consisted of a single component with an apparent molecular weight of 45 kDa, under reducing and non-reducing conditions (Figure 3A inset) suggesting a monomeric organization of the effector responsible of the hemagglutinating activity. The eluted fraction from affinity chromatography was applied to a HPLC size exclusion column and the obtained profile is shown in Fig. 3A. From the HPLC size exclusion step the purified lectin seems to have an approximate molecular weight of 43 kDa (Fig. 3B). The hemagglutinating activity of the purified fractions was maintained after 2 months at 20 °C, mildly affected when preincubated for 30 min at 50, 60 or 70 °C but reduced at 90 °C. Neither purified hemoagglutinin nor mucus showed agglutinating activity towards SRBC (Table 2). Table 2 Inhibition of hemagglutination activity activity of the S. spallanzanii mucus or isolated lectin with RE by various sugars Inhibitor Minimum concentration (mM) of sugar required for 100% inhibition of HA reaction Serum Isolated lectin D-galactose 25.0 mM 8.1 mM L-fucose 100.0 mM 16.5 mM D-Mannose 130 mM NI Rahmnose 130 mM NI Arabinose, cellobiose, D-glucose, Lactose, Lactulose, Maltose, Mannan, N-Ac-galactosamine N-Ac-glucosamine, D-raffinose; = no inhibition for 200 mM of sugar concentration 18 Fig. 2 Galactose-agarose affinity chromatography profile and eluted fractions from mucus. A: Diluted mucus was applied on a galactose-agarose column and elution step (1-20) performed with 100 mM galactose. The eluted fractions (11-14) show a protein concentration ranging from 0.08 to 0.18 mg/ml. B: Hemagglutinating activity (HA) against rabbit erythrocytes induced by: mucus (1), purified lectin fraction n. 26 (2), purified lectin fraction n. 27 (3), erythrocytes control (4) SsGBL bacterial agglutination The purified SsGBL agglutinated both Gram- positive and Gram-negative bacteria (Table 1, Fig. 4) and was inhibited by galactose (Table 2). Therefore, the SsGBL binding specificity varied significantly depending on the bacterial target used. In fact, it strongly agglutinated E. coli and V. alginolyticus, in a lesser extent A. hydrophyla and M. lysodeikticus (Table 1) whilst no agglutination was found by using S. aureus. Discussion Comparative immunology is important to understand a fundamental aspect of immunology particular for the phylogenetic perspective (Ballarin and Cammarata 2016) and the study of Annelids immunology have been deeply contributed (Engelmann et al., 2018). Lectins are important immunomediators in vertebrates and invertebrates (Kuhlman et al., 1989; Cooper et al., 1994; Matsushita et al., 1996; Tino and Wright, 1996; Odom and Vasta, 2006; Vasta, 2009). Many carbohydrate binding proteins have been already described in Annelida, both in marine and terrestrial species. Kawsar et al., (2010) purified a 32 kDa galactose-binding lectin from P. nuntia homogenates that showed hemagglutinating activity against human and rabbit erythrocytes. This lectin from P. nuntia revealed a clear antibacterial activity inhibiting the Gram-positive growth in vitro. Hirabayashi et al., (1998) isolated a 29 kDa lectin from L. terrestris body extracts. These carbohydrates-binding molecules are highly specific for sugar moieties. On account of their capability to bind carbohydrates involved in attachment of potential pathogens to host, lectins can protect the animal preventing its invasion from pathogens. Lectins are also involved in cell agglutination, recognizing structures on pathogens surface, they can opsonize them and enhance host phagocytic activity or activate the complement pathway (Matsushita et al., 1996; Cammarata et al., 2014). Due to these properties, lectins evidenced in the Table 3 Purification steps of SsGBL Purification stage Protein (mg) HA titer THA Specific activity THA/PC Purification (fold) Yield % Mucus 12.5 256 5120 410 1 100 Galactose-agarose 0.33 64 1280 3879 9.46 25 HPLC 0.015 8 160 10600 26 3 HA: hemagglutinating activity; THA: total hemagglutinating activity. 19 Fig. 3 (A) Affinity column purified fraction from galactose-agarose applied to a high-pressure liquid chromatography (HPLC) size exclusion column BioSuite 250-10 µm SEC 7,5 x 300 mm Waters, 350 psi pressure, 280 black line /254 red line nm (mAU). (A inset) SDS - PAGE (16%) of the purified lectins under reducing conditions; Standard proteins HPLC profile; Native size estimation of SsGBL by HPLC. The arrow points to the elution position of SsGBL. (B) Calibration standards (rhombus): phosphorylate b (97 kDa); bovine serum albumin (BSA, 67 kDa), enolase (46.7 kDa), myoglobin A (18.7 kDa) and RNaseA (13.7 kDa) body surface mucus can be considered potential antimicrobial agents. Among Annellida S. spallanzanii is one of the best known and abundant Mediterranean sabellid. In this animal, a large amount of mucus is secreted when specimens are subjected to different stress conditions leading to suppose its involvement as protective compartment against microorganisms and/or epibiosis (Stabili et al., 2011, 2014; Giangrande et al., 2014) as already observed in other invertebrates (Denny et al., 1989; Weis et al., 1998; Smith et al., 2010; Ogawa et al., 2011, Stabili et al., 2014). A defensive function in S. spallanzanii mucus was firstly suggested by Canicatti et al., (1992) who evidenced a haemolytic activity in this matrix. Recently, Stabili et al. isolated a lysozyme-like activity and an in vitro antimicrobial activity in S. spallanzanii mucus towards some Gram-negative bacteria (Stabili et al., 2009) clearly indicating the role of this compartment in defending the worms from bacterial attack serving as medium into which the antibacterial substances are exuded. Here a divalent-cation dependent lectin was newly discovered from mucus of sabellid, and we purified the SsGBL, a 43 kDa monomeric galactose-specific lectin from S. spallanzanii by using both affinity chromatography and high- pressure liquid chromatographic methods. Its agglutinating activity towards rabbit erythrocytes was significantly modified by the addition of calcium or EDTA. The activity shows good thermal stability, at temperature values comprised between 4°C and 37°C including the range of natural environment in which these annelid lives. Of particular interest, this lectin is a significant fraction (10-30%) of the soluble proteins of the S. Spallanzani mucus supporting an important functional role of this molecule. Galactose-binding lectins (GBLs) have been discovered and isolated also from others Annelids. Hirabayashi et al. (1998) first isolated from the earthworm, Lumbricus terrestris, a galactose- binding lectin of 29 kDa inhibited by a wide range of galactose-containing saccharides. The lectin is composed of two homologous domains of 14.5 kDa showing 27% identity among other GBLs and contained multiple short conserved motifs, "Gly-X-X- X-Gln-X-Trp". Another GBL with a molecular weight of 32 kDa was purified from the pacific annelid Perinereis nuntia ver. vallata by affinity chromatography showing a typical R lectin QxW sequence (Kwasar et al., 2010). In addition to the agglutinating activity against rabbit erythrocytes, the SsGBL bind both Gram- negative and Gram-positive bacteria; this ability to recognize and agglutinate exogenous target appears calcium dependent, according to the C-type lectins mode of action. 20 Fig. 4 The purified SsGBL displays the ability to agglutinate E. coli (A), V. alginolyticus (C) and M. lysodeikticus (E). E. coli (B), V. alginolyticus (D) and M. lysodeikticus (F) as negative control. BA: Bacterial agglutination In particular, the strongest agglutinating activity was observed towards the Gram-negative V. alginolyticus and E. coli present in coastal areas and in the harbors in which S. spallanzani lives, by contrast mucus agglutinated in a lesser extent A. hydrophyla and the Gram-positive M. lysodeikticus, but not against Staphylococcus aureus. These data correspond to the findings already recorded for the total mucus, suggesting that the SsGBL could represent an important effector responsible for the mucus defense role. The ability to recognize, bind and agglutinate bacteria has been well described among vertebrates in fish lectins (Bianchet et al., 2002; Odom and Vasta, 2006; Vasta et al., 2011), mammals (Cash et al., 2006; Vaishnava et al., 2011) and many invertebrates (Malagoli et al., 2006) suggesting an antibacterial activity (Matsui et al., 1994; Tateno et al., 2002). Among these, galactose-binding lectins have been described both in invertebrates and fish skin mucus, with bacterial agglutination properties. The skin mucus galectin from Japanese eel exhibited agglutination of Streptococcus difficile and E. coli (Suzuki et al., 2003). further confirming their protective role (Shiomi et al., 1989; Mistry et al., 2001; Suzuki et al., 2003; Ogawa et al., 2011). In 21 general galectins are parts of the fish defense system and mainly exist in organs and tissues that delineate the body from its surroundings, such as epidermal club cells of the skin, esophagus and gills (Nakamura et al., 2012). In this paper, we propose that the SsGBL is involved in the mucus body defense role, probably it is able to prevent bacterial invasion through the ability to agglutinate some bacteria strains. Our data suggest that the SsGBL could represent an important effector acting in contemporary presence with hemolytic factors and lysozyme as a first defense mechanism against potentially pathogens. Such synergic strategy is common and well known in invertebrates that lack acquired immunity and therefore reliant on mechanisms of innate immunity. This role is important taking into account that S. spallanzanii lives in eutrophic environments such as harbors where bacteria, including pathogens for humans, marine organisms, are abundant (Barg and Phillips, 1998). Although the worms may be aggressed by bacteria, they are able to survive bacterial attack. Unfortunately, by using the classical Edman degradation technique, N-terminal sequencing seem to be blocked. Microsequence analyses carried out by MALDI spectra, despite showing strong peaks didn’t give matches and the nLC-ESI MSMS only gave hits for keratin, and for the weaker sample a bit of BSA as well. The missing of significant match is plausible because the S. spallanzanii sequences are not well represented in the NCBI database and probably is not close enough in the NCBI database to give at least a match based on homology (data not showed). Even though our knowledge about composition, production and roles of mucus in various marine invertebrates remains incomplete, our results contribute to the understanding of the mucus protective properties in the investigated polychaete (Stabili et al., 2009; 2011). 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