Comparative aspect of proPO system in invertebrates ISJ 6: S67-S76, 2009 ISSN 1824-307X REVIEW The ascidian prophenoloxidase activating system M Cammarata, N Parrinello Marine immunobiology Laboratory, Department of Animal Biology, University of Palermo, Palermo, Italy Accepted March 13, 2009 Abstract Phenoloxidases/tyrosinases initiate melanin synthesis in almost all organisms, and are involved in different biological activities such as the colour change of human hair and the browning or blackening of fruit skin etc. In many invertebrates, defence reactions are linked to phenoloxidase activity and/or melanization. Contacts with foreign molecules are able to trigger the prophenoloxidase (proPO) system that requires serine protease cleavage for activating the zymogen to phenoloxidase (PO). It is generally accepted that the proPO system is fully expressed in arthropods, and, recently, progress in the regulation of crustacean and insect proPO activation steps have been achieved. After cells were stimulated by components of pathogen associated molecular pattern (PAMP), proPO activation takes place via zimogenic serine proteinase in turn activated by PAMPs followed by cascade, spatial and temporal control. The proPO activating system plays a defensive role in arthropods, molluscs, annelids, ascidians and the cephalochordate Branchiostoma belcheri. In the present paper, we report on ascidian proPO system and related molecules, with particular focus on the biochemical, cellular and molecular aspects of the Ciona intestinalis, proPO system of circulating hemocytes from naïve ascidians as well as of body wall following LPS inflammatory challenge. Key words: Ciona intestinalis; ascidians; proPO; phenoloxidase; immune response; hemocytes Phenoloxidases and related enzymes Melanin is a pigment ubiquitous throughout the animal kingdom. In invertebrates melanization is related to phenoloxidase (PO) and in part to tyrosinase, both are copper-dependent enzymes (monophenol, L-dopa; oxygen oxidoreductase; EC 1.14.18.1), that share similar active sites and catalyse the O-hydroxylation of monophenols (monophenoloxidase or cresolase activity) and the subsequent oxidation of the reaction products (o- diphenols) to quinones. Thus, substances forming copper ion complexes can be enzyme inhibitors (Kahn, 1985; Sugumaran et al., 1988). Tyrosine is the natural substrate of tyrosinase, which exhibits a lag–phase during the tyrosine conversion ascribed to an autocatalytic mechanism due to the production of L-dopa in the initial phase of the reaction pathway (Lerner, 1949). ________________________________________________________________________ Corresponding author: Matteo Cammarata Marine immunobiology Laboratory Department of Animal Biology University of Palermo via Archirafi, 90123 Palermo, Italy E-mail: camat@unipa.it Vertebrate tyrosinases form dimers whereas POs, only found in invertebrates, form oligomers, from monomers to pentamers. Although, both present two sites containing copper vary in their remaining sequence. In invertebrates, the prophenoloxidase (proPO) is converted to PO by proteolytic cleavage. The activation depends upon a cascade due to hemolymph proteases which are sensitive to peptidoglycans and lipopolysaccharides (LPSs) or other bacterial carbohydrates or fungal β-glucans. In crayfish hemocytes proPO is a 76 kDa glycoprotein that, after activation by β-1.3-glucans, is cleaved by specific serine proteases to produce the 60 kDa active PO (Aspan and Söderhäll, 1991). A similar cascade has been reported in other invertebrates (Beschin et al., 1998; Parrinello et al., 2001; Luna- Gonzales et al., 2003). Molecular analysis of PO and related protein POs can be sharply distinguished from tyrosinase and an independent evolution with short sequence traits conservation has been proposed. S67 Fig. 1 A. The Conserved Domain Architecture of C. intestinalis phenoloxidases performed by similarity searches of the NCBI Entrez Protein Database (CDART). B. Sequence alignments of C. intestinalis prophenoloxidases for comparison with arthropod POs, hemocyanins and various tyrosinases. The sequences shown are segments corresponding to the copper A and B binding sites of the hemocyanins. The homologous aa are shown in boldface, the three Histidine residues (H) participating in the Cu atom are shown in red. Conversely, a close similarity between arthropod phenoloxidases and hemocyanin, an oxygen carrier protein, has been shown (Söderhäll et al., 1996). Both proteins present sequence similarity and contain two oxygen-binding sites that reversibly bind two copper atoms (Decker and Tuczek, 2000), moreover hemocyanin-like proteins can act as phenoloxidases (Immesberger and Burmester, 2004). Like proPO, hemocyanins can be activated in vitro by SDS, trypsin and other effectors with denaturating property (Decker et al., 2001; Lee et al., 2004), or by chitin-binding antimicrobial peptides (Nagai et al., 2001). This activation has been imputed to the cleavage of crayfish hemocyanin subunit 2 at the N-terminal part (Lee et al., 2004). Decker et al. (2004) described similar results on the tarantula hemocyanin, and hypothesized that after N-terminal cleavage the hemocyanin active site becomes accessible also for phenolic substances. ProPOs from different arthropods have been cloned and sequenced (Aspan et al., 1995; Fujimoto et al., 1995; Hall et al., 1995; Kawabata et al., 1995). Comparative sequence analysis (Söderhäll et al., 1996), including several copper-containing proteins, showed that proPOs disclose sequence similarity to hemocyanins higher (49-59%) than to tyrosinases (33- 35 %) (Parrinello et al., 2003). S68 Table 1 Possible genes involved in the C. intestinalis proPO activated system CinPO-1 CinPO-2 Peroxinectin-like gene Cu-Zn SOD-like gene mRNA AJ7547813 AJ7547814 predicted XP_002126285 predicted XP_002122526 Protein Complete; 794 aa Partial at N-term; start ATG absent. 774 aa (768 aa in jgi:279870) Complete; 960 aa Complete; 154 aa putative size (kDa) 92.0 86.9 105.0 15.6 Domain/Binding sites Cu binding sites Cu binding sites Peroxidase/cell binding site Cu-Zn Reference Burmester et al 2003 Burmester et al 2003 Present paper Present paper Immesberger and Burmester (2004) cloned and sequenced two Ciona intestinalis PO cDNAs (CinPO-1 and CinPO-2). CinPO-1 and CinPO-2, with predicted molecular masses of 92.0 kDa and 86.9 kDa respectively, showed 43.2 % identity and do not contain signal peptides indicating a non classical release. Figure 1 shows the sequence alignment of C. intestinalis PO sites compared to arthopod POs, hemocyanins and some tyrosinases, and displays a close similarity between POs and hemocyanins. A third putative CinPO showing high similarity to CinPO-2 had been identified by a search on JGI Ciona intestinalis V2 as CinPO-3 (Cammarata et al., 2008). However, after a further analysis CinPO-3 appeared to be a product of an uncorrect assembly of the sequences in the V2 genome version. In fact, a long stretch of undetermined nucleotides, at the scaffold end, explained the assumed high similarity of introns and esons as well as of the presumptive protein sequences. ProPO activating system The first defence line of the innate immunity includes PO pathway products that participate in several responses such as melanization and encapsulation, cytotoxicity, phagocytosis, clotting, microbial killing and wound repair (Söderhäll, 1982; Cammarata et al., 1997; Gillespie et al., 1997; Huang et al., 2000; Nagai and Kawabata, 2000; Nappi and Ottaviani, 2000; Cerenius and Söderhäll, 2004; Jiravanichpaisal et al., 2006; Cerenius et al., 2008). It has been proposed that a molecular cross- talk takes place between the proPO system and cellular defence responses which share activation by microbial products signals, such as coagulation and blood cell degranulation (Lemaitre and Hoffmann 2007; Cerenius et al., 2008). Crustacean granular and semigranular hemocytes contain proPO, and, upon exposure to bacteria, they undergo degranulation in vitro leading to exocytosis (Johansson and Söderhäll, 1989b, 1999). In crayfish, Pacifastacus leniusculus, the 76- kDa peroxinectin, purified from the hemocytes (Johansson et al., 1995), mediates the attachment and spreading of hemocytes in vitro (Johansson and Söderhäll, 1988), and stimulates degranulation events when added to granular cells monolayer (Johansson and Söderhäll, 1989a, b). Peroxinectin- like is a cell adhesion protein also detected in shrimp (Poeneus monodon) hemocyte lysate supernatant (Sritunyalucksana et al., 2001). Sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69 %) and to various peroxidases or putative peroxidases from invertebrates and vertebrates. Since the biological effect of crayfish hemocyte peroxinectin is mimicked by the peptide Gly-Arg-Gly- Asp-Ser (GRGDS) (Johansson and Söderhäll, 1989c), the possibility exists that crayfish hemocyte integrin-like receptors recognize and bind RGD or KDG (Rouslahti, 1996; Holmblad et al., 1997). Vertebrate integrins form a family of integral membrane proteins that act in cell-cell adhesion and as receptors in trans-membrane signalling (Hines, 1992). Peroxinectin may act as opsonin, and promote the adhesion of bacteria or other particles to the phagocyte surface, facilitating their subsequent ingestion by the cell. Peroxonectin also binds Cu- Zn-superoxide dismutase (CuZnSOD) at the surface of circulating hemocytes, and this interaction, facilitated by the close localization, modulates both the enzyme activities. The hydrogen peroxide, produced by the superoxide dismutases, can be substrate for the peroxinectin and antimicrobical substances (Johansson et al., 1999). Therefore, the CuZnSOD is involved in arthropod proPO activating system. We carried out a bioinformatic analysis and, in Table 1, show for the first time that the C. intestinalis predicted peroxinectin-like gene contains both the active site of the peroxidase and the cell binding site (Gly-Arg-Gly-Asp-Ser, LKKGDR), moreover the deduced aminoacid complete sequence reveals 35 % identity with P. leniulusculus peroxinectin. In C. intestinalis genome, the presence of eleven alpha and five β chain integrin genes, suggest putative peroxinectin cell surface S69 Table 2 Properties and modulation of the ascidian phenoloxidases C. intestinalis S. plicata B.schlosseri P. mamillata H. roretzi References (1-7) (5,7,8) (9,10) (5,11) (12) Treatment HLS THS HLS Trypsin Trypsin + STI ----- ----- ----- ----- ----- LPS PO inhibitors Calcium effect No No No Yes No No PO Containing cell types URG Granular amebocytes Morula Morula compartment cell granular hemocytes ND PO Subunit MW 74 90 ND 80 70 62 Method Cloned HLS Isolated HLS Isolated Biological activities Cytotoxicity Cytotoxicity Non fusion reaction ND Antimicrobial activity Hemocyte lysate supernatant (HLS); Tunic homogenate supernatant (THS); Unilocular refractile granulocytes(URG) (1) Söderhäll and Smith, 1992; (2) Peddie and Smith, 1993; (3) Parrinello et al., 1995; (4) Cammarata et al., 1996; (5) Parrinello et al., 2003; (6) Cammarata et al., 2008; (7) Arizza et al., 1995; (8) Cammarata et al., 1997; (9) Ballarin et al., 1994; (10) Ballarin et al., 2008; (11) Cammarata et al., 1999; (12) Hata et al., 1998 adhesion receptors (Ewan et al., 2005). In Table 1, we also report the sequence of a C. intestinalis putative gene Cu-Zn SOD with 46 % identity to the predicted aminoacid sequence of the H. roretzi enzyme, obtained by using the aminoacid sequence of H. roretzi Cu-ZnSOD (Abe et al., 1999) as a query in an aminoacid-based BLAST search (tblastn) versus the NCBI/GenBank database. Ascidian proPO activating system and innate immunity Ascidians occupy a key phylogenetic position in the evolutionary line leading to vertebrates (Hori and Osawa 1987; Field et al., 1988; Zeng and Swalla 2005; Delsuc et al., 2006), therefore both solitary and colonial ascidians are of interest in studying the evolution of defence mechanisms. Phagocytosis, cytotoxicity, encapsulation and tissue damage (Wright and Cooper, 1983; Parrinello and Patricolo, 1984; Parrinello et al., 1984, 2001, 2007; Ballarin et al., 2008) in inflammatory responses, as well as in inflammatory events linked to allorecognition responses, have been shown (Sabbadin, 1982; Rinkevich, 1992; Raftos et al., 1988). Both share hemocytes degranulation in tissues of solitary ascidians (Parrinello et al., 1984) and in the contact area of incompatible botryllid colonies (Sabbadin, 1982; Ballarin, 2008). Differently than arthropod POs, which are monophenoloxidases, ascidian hemocyte POs are orthodiphenoloxidases. O-diphenol oxidase activity and phenolic compounds were at first identified by histochemical reaction (Barrington and Thorpe, 1968) in the tunic hemocytes suggesting a quinone- tanning system involved in the production of tunic scleroprotein (Chaga, 1980). Although ascidian POs show the highest activity at 6-9 pH range (Jackson et al., 1993; Arizza et al., 1995; Ballarin et al., 1994), they can differ in several biochemical properties. In Botryllus schlosseri the L- dopa oxydizing activity is enhanced by divalent cations (Ballarin el al., 1994), whereas the PO activity of other ascidian species does not appear to be Ca2+ or Mg2+-dependent (Jackson et al., 1993; Arizza et al., 1995; Cammarata et al., 1999). In crustaceans, although calcium ions are requested, a high cation concentration exerts a suppressive effect. A further difference concerns the activating mechanism: β 1,3-glucans and oligosaccharides induce proPO-activation in arthopods (Söderhäll and Smith, 1986; Söderhäll, 1992) and in solitary ascidian C. intestinalis (Jackson et al., 1993), whereas do not activate Styela plicata (Arizza et al., 1995) and B. schlosseri (Ballarin et al., 1994) POs. Ascidian orthodiphenoloxidases are copper- dependent enzymes inhibitable by copper chelating substances (Kahn, 1985; Sugumaran et al., 1988). Like in arthropods, the ascidian proPO requires proteolytic cleavage for its activation. The level of PO activity is significantly higher after incubation with serin-proteases, decreases as an effect of protease inhibitors, and it is activated by LPS (Table 2). Smith and Peddie (1992) and Jackson et al. (1993) reported that the LPS-sensitive protease activity, contained in the C. intestinalis hemocyte lysate supernatants, may be associated with in vivo prophenoloxidase activaction. Benzamidine, soybean trypsin inhibitor (STI), phenylmethylsulphonyl fluoride, tosyl phenylalanyl S70 chloromethyl ketone, tosyl-L-lysin-chloromethyl ketone inhibited both protease and proPO activation, this inhibitory activity is short lived and precedes hemocyte PO activity (Jackson and Smith, 1993). At least three proteases are contained in solitary ascidians C. intestinalis, S. plicata (unpublished data) and Phallusia mamillata hemocytes (Guerrieri et al., 2000), and could regulate the otherwise uncontrolled protease activity. PO containing hemocytes Phenoloxidase participates in tunic formation (Chaga, 1980), melanin production and non fusion reaction (Ballarin et al., 1996), and an increased PO activity can be found in C. intestinalis tissues inflamed by LPS inoculation (Parrinello et al., 2001). In C. intestinalis (Smith and Söderhäll, 1991) and B. schlosseri (Ballarin et al., 1993) PO oxidative activity of circulating hemocytes from naïve ascidians as well as hemocyte lysate supernatants has been revealed by L-dopa reaction. A similar activity has also been reported in Ascidia mentula, Ascidia virginea, Ascidiella scabra, Ascidiella aspersa, Polycarpa pomaria, Dendrodoa grossularia and Morchellium argus (Jackson et al., 1993). The specificity of the PO reaction of S. plicata, P. mamillata and C. intestinalis hemocytes (Arizza et al., 1995; Parrinello et al., 2003) has been supported by tropolone, phenylthiourea and diethyltiocarbamate usually used as inhibitors (Sugumaran et al., 1988; Cadenas, 1989; Kahn, 1995). The enzymatic assay of the hemocyte lysate and the cytochemical reaction of the hemocytes revealed a limited heterogeneity in the ascidian PO- containing cell types. PO-activity as well as the possible substrates tunichrome and halocyamine- reducing polyphenol, have mainly been identified in the hemocytes called "morula cells" (Azumi et al., 1990; He et al., 1992; Ballarin et al., 1996; Parrinello et al., 2003). Evidence of PO positive “morula cells” have also been drawn by assaying hemocyte populations enriched through a density gradient separation of the hemolymph from C. intestinalis (Jackson et al., 1993; Parrinello et al., 2003), B. schosseri (Ballarin et al., 1994) and S. plicata (Arizza et al., 2005). This hemocyte-type is a round globular granulocyte (berry-like under the light microscope; 5.5-11.0 µm diameter) with large granules containing material of various electron- density (De Leo, 1992). Light microscopy observations show large globular granules varying in number, shape and content feature. Although Smith and Peddie (1992) suggested that C. intestinalis morula cells contain PO, we found a weak reaction after the treatment with L-dopa-MBTH. On the contrary, a strong PO activity was found in the unilocular refractile granulocytes (URGs) identified in the hemolymph and characterized by a single PO- positive large granule that occupies the largest part of the cytoplasm (Parrinello et al., 2003). In addition PO-positive large granules were also found in granulocytes. Although a defined differentiation line was not established, the possibility exists that granulocytes with large granules, URGs and morula cells may be components of a lineage characterized by a different state of the granule content including PO activity level as well as hemocyte functions. Accordingly, URGs exert PO-dependent cytotoxic activity whereas morula cells do not show any cytotoxic activity against erythrocytes (Parrinello et al., 1996) and tumour cell lines (Peddie and Smith, 1993). In P. mamillata, “hemocytes with large granules” show a low PO activity whereas the morula cells do not show any positive reaction with L-dopa-MBTH. The activity of the hemocyte lysate supernatant of enriched populations, obtained through a Percoll density gradient, can be enhanced by trypsin that presumably activates the proPO remaining after cell lysis. Usually, hemocytes preparation and further treatments may cause the activation of a part of proPO content by endogeneous proteases. In this ascidian, another hemocyte type named compartment cells characterized by few large vacuoles show spontaneous proPO activation, and vacuoles appear to be PO-positive. However, the lysate supernatant from enriched populations are less sensitive to trypsin proteolysis activation indicating that these cells may be more reactive to handling which can activate proPO. Proteolytic activation of proPO is sustained by electrophoresis and L-dopa-MBTH stain of trypsin-treated and untreated hemocytes with large granule lysate supernatants; the electropherograms show an increased mobility of the enzyme after proteolysis and, accordingly, reveal an additional protein fragment (Parrinello et al., 2003). Tunic POs and inflammatory response Recently, the PO activity of tunic homogenate supernatant (THS) from naïve C. intestinalis has been assayed (Cammarata et al., 2008). As already reported upon the hemocyte lysate supernatant (HLS), the PO activity of THS is Ca2+-independent, but, unlike HLS (pH 6-9) requires a lower pH (7-8) and is more thermo-stable. The THS activity is lost after two days at 0 °C and after about one year at - 80 °C, whereas the HLS activity disappears after 2-3 h at 0 °C and 3-4 week at -80 °C. Likewise the HLS- proPO, the treatment with exogenous trypsin enhances the activity and STI inhibits it, whereas a further difference resulted from a more effective activation due to LPS acting at a lower concentration than that needed for activating HLS-proPO. In addition, since LPS inoculation enhances the THS- PO activity of samples assayed in the absence of trypsin the possibility exists that in the tunic several serine proteases or different proteases could be involved in the activation phase (Cammarata et al., 2008). To check for tunic matrix and tunic cells PO activity in the inflammatory response, an in vitro enzyme assay of the inflamed tunic tissue excised at 24 or 48 h after LPS inoculation has been performed. The PO activity appears to be distributed throughout the tunic matrix, as well as inside the large granule of URGs and hemocytes with large granules. These observations are in accordance with the report on circulating hemocytes from naïve ascidians, whereas it is of interest that morula cells in the inflamed tunic disclose an evident PO activity suggesting a distinct step of the cell lineage not found in circulating hemolymph from naïve ascidians S71 and presumably due to the LPS challenge. Microscope observations of wet inflamed tunic fragments after L-dopa-MBTH treatment, showed both a strong PO reaction and a high density of PO- positive hemocyte populations (Fig. 2). Since the deduced aminoacid sequence of a C. intestinalis CinPO-2 isoform has been reported (GenBank Accession n. AJ547814) by Immersberg and Burmester (2004), a peptide (11-aa, EFHNDRRNRGF) has been selected through an antigen-prediction program, and anti-CinPO-2- peptide specific antibodies has been raised in rabbits (Cammarata et al., 2008). The immunohistochemistry reaction shows an intense dye of the inflammatory cells supporting that proPO- 2 synthesis could be enhanced by LPS inoculation (Cammarata et al., 2008). The same assay revealed that, after LPS inoculation, the enzyme is distributed in the tunic matrix, mainly in the outer layer, in addition anti-CinPO-2 antibodies also marked the pharynx vessel epithelia. Immesberger and Burmester (2004) reported that 86.9 kDa is the predicted molecular size of CinPO-2, and this value fits with the 90kDa revealed by the L-dopa-MBTH reaction and immunoblotting analysis of THS from naïve ascidians (Cammarata et al., 2008). After the LPS inoculation, an additional 120 kDa band reacts with L-dopa-MBTH and anti- CinPO2 antibodies, whereas a 170 kDa L-dopa- MBTH positive band does not react with the antibodies. Presumably, this band could be considered as a dimeric form of CinPO-1 (92.0 kDa) which cannot be identified by antibodies directed to a CinPO-2 peptide. Anyway, an oligomerisation process may be responsible of CinPOs higher in size (120 and 170 kDa). A similar process may be taken in account for the differences observed in the THS- and HLS-PO (Parrinello et al. 2003) molecular sizes, and suggests that further analyses are needed. Biological activity of PO and related molecules In invertebrates, substances with cytotoxic activity include molecular derivates of oxygen and nitrogen, antimicrobial peptides, lectins and quinoid intermediates of melanin (Parrinello 1996; Nappi and Ottaviani 2000). The oxygen reactive forms, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide anions have been implicated as components of the cytotoxic mechanisms of vertebrates (Cadenas, 1989) and invertebrates (Bell and Smith, 1993; Anderson, 1994; Valembois and Lassegues, 1995; Nappi and Ottaviani 2000). The propensity of quinones for redox-cycling makes these eumelanin precursors potential sources of reactive forms of oxygen (Riley, 1988; O'Brien, 1991). Indeed, it has been demonstrated (Smit et al, 1996) that phenolic compounds, converted into toxic products by tyrosinase, exhibit cytotoxic activity towards human melanoma cells. We reported that the morula cells form S. plicata (Cammarata et al., 1997) and URGs from C. intestinalis (Parrinello et al., 1995) display PO- dependent cytotoxic activity against tumour cell lines (unpublished data) and rabbit erythrocytes due to quinones which are known to be cytotoxic (Pawelek and Lerner, 1978; Cotelle et al., 1991; Fu et al., 1994; Parrinello et al., 2003).The presence of quinones, possibly originated by PO-driven tunichrome oxidation, has been reported in ascidians. Differently, ROI derived from the PO pathway are cytotoxic factors in B. schosseri non- fusion alloreaction (Ballarin et al., 2002, 2008). Indeed, in vivo, quinones could be activated by enzymatic reduction as, under aerobic conditions, the semiquinone radical autoxidizes and forms superoxide anion radicals (Nappi and Vass, 1993). The superoxide anion, hydrogen peroxide and trace amounts of transitional-metal ions form a hydroxyl radical (Fenton reaction), which is the most toxic of all oxygen products. There is only indirect evidence that, in C. intestinalis, proPO-activation products have a stimulatory influence on hemocyte behaviour in vitro. In this ascidian, the crude hemocyte lysate supernatant, which contains active PO and LPS- sensitive proteases, has a marked opsonic influence on the uptake of bacteria by phagocytic amoebocytes (Smith and Peddie, 1992). Such opsonic potential match to the degree of the enzyme activity; in addition, PO and proteases inhibition with benzamidine or STI precludes the opsonic effect, while pre-treatment of the lysate supernatants with LPS produces a greatly elevated phagocytic response (Smith and Peddie, 1992). Figure 3 shows a tentative model of the C. intestinalis proPO activating system, based on present knowledge. Finally, morula cells can release proPO-activation products for tunic formation and regeneration in Goniocarpa Fig. 2 Tunic fragment of C. intestinalis 24 h treated with L-dopa-MBTH after the inoculation of LPS. Arrowhead indicates the PO reaction at the injection site. Bar (A) = 1 cm; (B) = 200 µm. S72 Fig. 3 A model for the activation and modulation of the pro-PO system in C. intestinalis, based on the following reference results: Söderhäll and Smith, 1992; Peddie and Smith, 1993; Parrinello et al., 1995; Cammarata et al., 1996, 1997; Parrinello et al., 2003; Cammarata et al., 2008. rustica, Halocynthia aurantium (Chaga, 1980) and B. schlosseri (Zaniolo, 1981), and can be involved in the formation of clotting (Wright, 1981), and in the encapsulation of foreign particles (Anderson, 1971; Parrinello, 1996). Conclusions The melanogenetic pathway can be dependent on tyrosinase or phenoloxidase activity, the proPO activating system appears to be a sophisticated system which represents an evolutionary independent defence mechanism characteristic of invertebrates. Apparently protostomes and deuterostomes share proPO and PO-related factors and activities. Phenoloxidase activity and proPO-like components have been reported in the hemolymph of arthropods, annelids, molluscs, echinoderms and cephalocordata (Roch et al., 1992; Nappi and Vass, 1993; Beschin et al., 1998; Luna-Gonzales et al., 2003; Pang et al., 2005). Preliminary sequence analysis suggest that hemocyanin could be the evolutionary ancestor of PO and a parallel molecular evolution cannot be excluded. Taking in account the C. intestinalis inflammatory response challenged by LPS, the proPO system appears to be a component (Cammarata et al., 2008) of a very complex reaction that presumably involves several interacting cell types and factors, namely inducible cytokine-like molecules (Parrinello et al., 2007), CiFACIT- collagen (Vizzini et al., 2007), and CiTNFα (Parrinello et al., 2008). The LPS or glucans activating effect could be associated with one or more serine proteases modulated by trypsin inhibitors, Cu-ZnSOD and peroxinectin-like molecules. Therefore, LPS- and glucan-binding proteins appear to be suitable candidates as recognition molecules central to several defence reactions. Although some insight has been gained into the invertebrate proPO system, host recognition proteins, biological activities, molecular interaction, and signal transduction pathways, little is known on the ascidian cascade. Acknowledgements This work was supported by a MIUR (COFIN2006 n. 2006059857) research grant. References Abe Y, Ishikawa G, Satoh H, Azumi K, Yokosawa H. Primary structure and function of superoxide S73 dismutase from the ascidian Halocynthia roretzi Comp. Biochem. Physiol. 122B: 321-326, 1999. Anderson RS. Cellular responses to foreign bodies in the tunicate Molgula manhattensis. Biol. Bull. 141: 91-98, 1971 Anderson RS. Hemocyte-derived reactive oxygen intermediate production in four bivalve mollusks. Dev. Comp. Immunol. 18: 89-96, 1994. Arizza V, Cammarata M, Tomasino MC, Parrinello N. Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidians Styela plicata. J. Invertebr. Pathol. 66: 297-302, 1995. Aspán A, Huang TS, Cerenius L, Söderhäll K. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. USA 92: 939-943,1995. Aspán A, Söderhäll K. Purification of prophenoloxidase from crayfish blood cells and its activation by an endogenous serine protease. Insect Biochem. 21: 363-373, 1991. Azumi K, Yokosawa H, S Ishii. Presence of 3-4 dihydroxyphenylalanine-containing peptides in hemocytes of the ascidian, Halocynthia roretzi. Experientia. 46: 1020-1023, 1990. Ballarin L, Cima F, Floreani M, Sabbadin A. Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri. Comp. Biochem. Physiol. 133C: 411-4118, 2002. Ballarin L, Cima F, Sabbadin A, Morula cells and histocompatibility in the colonial ascidian Botryllus schlosseri. Zool. Sci. 12: 757-764, 1996. Ballarin L, Cima F, Sabbadin A. Phenoloxidase in the colonial ascidian Botryllus schlosseri (Urochordata, Ascidiacea). Anim. Biol. 3: 41- 48, 1994. Ballarin L, Cima F, Sabbadin A. Histochemical staining and characterization of the colonial ascidian Botryllus schlosseri hemocytes. Boll. Zool. 60: 19-24, 1993. Ballarin L. Immunobiology of compound ascidians, with particular reference to Botryllus schlosseri: state of art. Inv. Surv. J. 5: 54-74, 2008 Barrington EJW, Thorpe A. Histochemical and biochemical aspects of iodine binding in the tunic of ascidian Dendrodoa grossularia. Proc. Roy. Soc. B171: 91-109, 1968. Bell K, Smith VJ. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.) Dev. Comp. Immunol. 17: 211- 219,1993. Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, et al. Identification and cloning of a glucan- and lipopolysaccharide- binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J. Biol. Chem. 273: 24948-54, 1998. Cadenas E. Biochemistry of oxygen toxicity. Ann. Res. Biochem. 58: 79-110, 1989. Cammarata M, Arizza V, Cianciolo C, Parrinello D, Vazzana M, Vizzini A, et al., The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis.Cell Tissue Res. 333: 481-492, 2008. Cammarata M, Arizza V, Parrinello N, Candore G, Caruso C. Phenoloxidase-dependent cytotoxic mechanism in ascidian (Styela plicata) hemocytes active against erythrocytes and K562 tumor cells. Eur. J. Cell Biol. 74: 302-307, 1997. Cammarata M, Arizza V, Savona B, Vazzana M, Parrinello D. Prophenoloxidase in the hemocyte of Phallusia mamillata Anim. Biol. 8: 15-17, 1999. Cammarata M, Arizza V, Vazzana M, Parrinello N, Prophenoloxidase activating system in tunicate hemolymph. It. J. Zool. 63: 345-351, 1996. Cerenius L, Söderhäll K. The prophenoloxidase- activating system in invertebrates Immunol. Rev. 198: 116-126, 2004. Cerenius L, Lee BL, Söderhäll K. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 29: 263-271, 2008. Chaga OY. Ortho-diphenoloxidase system of Ascidians. Tsitologia 22: 619-625, 1980. Cotelle N, Moreau S, Cotelle P, Catteau JP, Bernier JL, Henicard. Generation of free radicals by simple prenylated hydroquinone derivativs, natural antitumor agents from the marine urochordate Aplidium californicum. Chem. Res. Toxicol. 4: 300-305, 1991. De Leo G. Ascidian hemocytes and their involvement in defence reactions. Boll. Zool. 59: 195-213, 1992. Decker H, Jaenicke E. Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Dev. Comp. Immunol. 28: 673- 687, 2004. Decker H, Ryan M, Jaenicke E, Terwilliger N. SDS- induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister. J. Biol. Chem. 276: 17796-17799, 2001. Decker H, Tuczek F.Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trends Biochem. Sci. 25: 392-397, 2000. Delsuc F, Brinkmann H, Chourrout D, Philippe H. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439: 965-968, 2006. Ewan R, Huxley-Jones J, Mould AP, Humphries MJ, Robertson DL, Boot-Handford RP. The integrins of the urochordate Ciona intestinalis provide novel insights into the molecular evolution of the vertebrate integrin family. BMC Evol. Biol. 5:31 2005. Field KG, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselin MT, et al., Molecular phylogeny of the animal kingdom Science 239: 748-753, 1988. Fu X, Hossain MB, Van der Helm D, Schmitz FJ, Longithorone A. Unprecedented dimeric prenylated quinone from the tunicate Aplydium longithorax. J. Amer. Chem. Soc. 116: 12125- 12126, 1994. Fujimoto K, Okino N, Kawabata S, Iwanaga S, Ohnishi E. Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster. Proc. Natl. Acd. Sci. USA 92: 7769-7773, 1995. Guerrieri N, Scippa S, Maietti M, De Vincentiis M, Cerletti. Protease activity in fractionated blood S74 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Arizza%20V%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus http://www.ncbi.nlm.nih.gov/pubmed/16495997?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum http://www.ncbi.nlm.nih.gov/pubmed/16495997?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Ohnishi%20E%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus cells of the vanadium accumulating ascidian Phallusia mammillata. Comp. Biochem. Physiol. 125A : 445-450, 2000. Hall M, Scott T, Sugumaran M, Söderhäll K, Law J. Prophenoloxidase of the hawkmoth, Manduca sexta. Purification, activation, substrate specificity of the active enzyme, and molecular cloning. Proc. Natl. Acad. Sci. USA 92: 7764- 7768, 1995. Hata S, Azumi K, Yokosawa H. Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles. Comp. Biochem. Physiol. 119B: 769- 776, 1998. He X, Kustin K, Parry DL, Robinson WE, Ruberto G, Nakanishi K. In vivo incorporation of 14C- phenylalanine into ascidian tunichrome. Experientia 48: 367-371, 1992. Holmblad T, Thörnqvist PO, Söderhäll K, Johansson MW. Identification and cloning of an integrin beta subunit from hemocytes of the freshwater crayfish Pacifastacus leniusculus. J. Exp. Zool. 277: 255-261, 1997. Huang TS, Wang H, Lee SY Johansson MW, Söderhäll K, Cerenius L. A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade. J. Biol. Chem. 275: 9996-10001, 2000. Immesberger A, Burmester T. Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily. J. Comp. Physiol. [B] 174: 169-180, 2004. Jackson AD, Smith VJ, Peddie CM. In vitro phenoloxidase activity in the blood of Ciona intestinalis and other ascidians. Dev. Comp. Immunol. 17: 97-108, 1993. Jackson AD, Smith VJ. LPS-sensitive protease activity in the blood cells of the solitary ascidian, Ciona intestinalis (L) Comp. Biochem. Physiol. 106B: 505-512, 1993. Jiravanichpaisal P, Lee BL, Söderhäll K. Cell- mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization. Immunobiology 211: 213-236, 2006 Johansson MW, Holmblad T, Thörnqvist PO, Cammarata M, Parrinello N, Söderhäll K. A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish. J. Cell Sci. 112: 917- 925, 1999. Johansson MW, Lind MI, Homblad T, Thörnqvist PO, Söderhäll K. Peroxinectin, a novel cell adhesion protein from crayfish blood. Biochem. Biophys. Res. Comm. 216: 1079-1087, 1995. Johansson MW, Söderhäll K. A cell adhesion factor from crayfish hemocytes has degranulating activity towards crayfish granular cells. Insect Biochem.19: 183-190, 1989a. Johansson MW, Söderhäll K. Cellular immunity in crustaceans and proPO system. Parasitol. Today 5: 171-176, 1989b. Johansson MW, Söderhäll K. A peptide containing the cell adhesion sequence RGD can mediate degranulation and cell adhesion of crayfish hemocytes in vitro. Insect Biochem.19: 573- 579, 1989c. Johansson MW, Söderhäll K. Isolation and purification of a cell adhesion factor from crayfish blood cells. J. Cell Biol. 106: 1795- 1803, 1988. Kahn V. Tropolone a compound that can aid in differentiating between tyrosinase and peroxidase. Biochemistry 24: 915-920, 1985. Kawabata T, Yasuhara Y, Ochiai M, Matsuura S, Ashida M. Molecular cloning of insect pro- phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin. Proc. Natl. Acad. Sci. USA 92: 7774-7778, 1995. Lee SY, Lee BL, Söderhäll K. Processing of crayfish hemocyanin subunits into phenoloxidase. Biochem. Biophys. Res. Com. 322: 490-496, 2004. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25: 697-743, 2007. Lerner AB. On the metabolism of phenylalanine and tyrosine. J. Bio. Chem.181: 281-294, 1949. Luna-Gonzalez A, Maeda-Martınez AN, Vargas- Albores F, Ascencio-Valle F, Robles-Mungaray M. Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs Fish Shellfish Immunol. 15: 275-282, 2003. Nagai T, Kawabata S. A link between blood coagulation and prophenoloxidase activation in arthropod host defense. J. Biol. Chem. 275: 29264-29267, 2000. Nagai T, Osaki T, Kawabata S. Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J. Biol. Chem. 276: 27166-27170, 2001. Nappi AJ, Ottaviani E. Cytotoxicity and cytotoxic molecules in invertebrates. BioEssays 22: 469- 480, 2000. Nappi AJ, Vass E. Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res. 6: 117-126, 1993. O'Brien PJ. Molecular mechanisms of quinone cytotoxicity. Chem. Biol. Interact. 80: 1-41, 1991. Pang Q, Zhang S, Wang C, Shi X, Sun Y. Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense Fish Shellfish Immunol. 17: 477-487, 2004. Parrinello N, Arizza V, Cammarata M, Giaramita FT, Pergolizzi M, Vazzana M, et al., Inducible lectins with galectin properties and human IL1α epitopes opsonize yeasts in the ascidian Ciona intestinalis inflammatory response. Cell Tissue Res. 329: 79-90, 2007. Parrinello N, Arizza V, Chinnici C, Parrinello D, Cammarata M. Phenoloxidases in ascidian hemocytes: characterization of the pro- phenoloxidase activating system. Comp. Biochem. Physiol. 135B: 583-591, 2003. Parrinello N, Cammarata M, Arizza V. Univacuolar refractile hemocytes from the tunicate Ciona intestinalis are cytotoxic for mammalian erythrocytes in vitro. Biol. Bull. 190: 418-425, 1996. S75 javascript:AL_get(this,%20'jour',%20'J%20Exp%20Zool.'); javascript:AL_get(this,%20'jour',%20'Fish%20Shellfish%20Immunol.'); Parrinello N, Cammarata M, Lipari L, Arizza V. Sphingomyelin inhibition of Ciona intestinalis (Tunicata) cytotoxic hemocytes assayed against sheep erythrocytes. Dev. Comp. Immunol. 19: 31-41, 1995. Parrinello N, Cammarata M, Vazzana M, Arizza V, Vizzini A, Cooper EL In: Yokosawa H, Lambert CC (eds), Immunological activity of ascidian hemocytes. The Biology of Ascidians, Springer-Verlag, Tokyo, pp 395-401, 2001. Parrinello N, Patricolo E, Canicattì C, Inflammatory like reaction in the tunic of Ciona intestinalis (Tunicata) I. Encapsulation and tissue injury. Biol. Bull. 167: 229-273, 1984. Parrinello N, Patricolo E. Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). II. Capsule components Biol. Bull. 167: 238-250, 1984. Parrinello N, Vizzini A, Arizza V, Salerno G, Parrinello D, Cammarata M, et al. Enhanced expression of a cloned and sequenced Ciona intestinalis TNFalpha-like (CiTNF alpha) gene during the LPS-induced inflammatory response. Cell Tissue Res. 334: 305-317, 2008. Parrinello N. Cytotoxic activity of tunicate hemocytes. In: Rinkevich B, Müller WEG (eds), Invertebrate immunology, Springer, Berlin, pp 190-217, 1996. Pawelek JM, Lerner AB. 5,6-Dihydroxyindol is a melanin precursor showing potent cytotoxicity. Nature 276: 627-628, 1978. Peddie CM, Smith VJ. In vitro spontaneous cytotoxic activity against mammalian target cells by the hemocytes of the solitary ascidian, Ciona intestinalis. J. Exp. Zool. 267: 616-623, 1993. Raftos DA, Briscoe DA, Tait NN. The mode of recognition of allogeneic tissue in the solitary urochordate Styela plicata. Transplantation 45: 1123-1126, 1988. Riley PA. Radicals in melanin biochemistry. Ann. NY Acad. .Sci. 551: 111-120, 1988. Rinkevich B. Aspects of incompatibility nature in botryllid ascidians. Anim. Biol. 1: 17-28, 1992. Roch P, Canicattì C, Sammarco S. Tetrameric structure of the active phenoloxidase evidenced in the coelomocytes of the echinoderm Holothuria tubulosa. Comp. Biochem. Physiol. 102B: 349-355, 1992. Ruoslahti E. Integrin signalling and matrix assembly. Tumour Biol. 17: 117-124, 1996. Sabbadin A. Formal genetics of ascidians. Amer. Zool. 22: 765-777, 1982. Smit NPM, Peters K, Menko W, Westerhof W, Pavel S, Riley PA. Cytotoxicity of a selected series of substituted phenols towards cultured melanoma cells. Melanoma Res. 2: 295-304, 1996. Smith VJ, Peddie CM. Cell cooperation during host defence in the solitary tunicate Ciona intestinalis (L.). Biol. Bull. 116: 211-219, 1992. Smith VJ, Söderhäll K. A comparison of phenoloxidase activity in the blood of marine invertebrates. Dev .Comp. Immunol. 15: 251-261, 1991. Söderhäll K, Cerenius L. Crustacean Immunity. Annu. Rev. Fish Dis. 3-23, 1992. Söderhäll K, Smith VJ. The prophenoloxidase activating system as a recognition and defence system in arthropods. In: Gupta AP (ed), Hemocytic and humoral immunity in arthropods, Wiley, New York, pp 251-286, 1986. Söderhäll K. Biochemical and molecular aspects of cellular communication in arthropods. Boll. Zool. 59: 141-151, 1992. Söderhäll K. Molecular analysis of the prophenoloxidase activating system In: Söderhäll K, Iwanaga S, Vasta G (eds), New directions in invertebrate immunology, SOS Publications , NJ, pp 229-253, 1996. Söderhäll K. Prophenoloxidase activating system and melanization- a recognition mechanism of arthopods? - A review. Dev. Comp. Immunol. 6: 601-611, 1982. Sritunyalucksana K, Wongsuebsantati K, Johansson MW, Söderhäll K. Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon. Dev. Comp. Immunol. 25: 353-363, 2001. Sugumaran M, Hennigan B, Semensi V, Mitchell M, Rivera T. Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinefrine by cuticular phenoloxidase from Sarcophaga bullata. Arch. Insect Biochem. Physiol. 8: 229-241, 1988. Valembois P, Lassegues M, In vitro generation of reactive oxygen species by free coelomic cells of the annelid Eisenia fetida andrei: An analysis by chemioluminescence and nitro blue tetrazolium reduction. Dev. Comp. Immunol. 19: 195-204, 1995. Vizzini A, Pergolizzi M, Vazzana M, Salerno G, Di Sano C, Macaluso P, et al. FACIT collagen (1alpha-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis. Dev. Comp. Immunol. 32: 682-692, 2008. Wright RK, Cooper EL. Inflammatory reactions of the protochordata. Amer. Zool. 23: 205-211, 1983. Wright RK. Urochordates. In: Invertebrate blood cells. Ratcliffe NA, Rowley AF (eds) Vol. 2, Academic Press, London, 1981. Zaniolo G. Histology of the ascidian Botryllus schlosseri tunic: In particular, the test cells. Boll. Zool. 48: 169-178, 1981. Zeng L, Swalla BJ. Molecular phylogeny of the protochordates: chordate evolution Can. J. Zool. 83: 24-33, 2005. S76 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Raftos%20DA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Briscoe%20DA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Tait%20NN%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus