Phenoloxidases and cytotoxicity in ascidians: an overview

Phenoloxidases (POs) belong to a family of copper-containing proteins (including also hemocyanins) widely distributed among invertebrates. They are able to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterize the nonfusion reaction of botryllid ascidians. Expressed genes for putative POs have been recently identified in the solitary ascidian C. intestinalis (CiPO1 and CiPO2) and the compound ascidian Botryllus schlosseri. Multiple sequence alignments evidenced the similarity between the sequences of ascidian POs and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins, which share common precursors with arthropod proPOs. Botryllus and Ciona POs grouped in the same cluster, and all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the Botryllus PO is transcribed inside morula cells, a characteristic cytotoxic haemocyte type.