Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily.

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157-2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.