Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri

Phenoloxidases (POs) constitute a family of copper-containing enzymes widely distributed among invertebrates. They exert a pivotal role in immune defences as they induce cytotoxicity through the conversion of phenols to quinones and the production of reactive oxygen species. 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 characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between 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. Ascidian POs and arthropod proPOs grouped in the same cluster and 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. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypotheses for the regulation of enzyme activity.