Calbindin-D32k in the Echinoderm Nervous System connective tissue plexus. (M–O) Anti-calbindin 1 (M), anti-parvalbumin (N), and anti-calbindin 2 (O) immunoreactivity was also present in the nerve plate of the tube feet’s disk. Al, ambulacral lumen; ctp, connective tissue plexus; icc, inner cluster of cells; me, mesothelium; np, nerve plate; occ, outer cluster of cells. doi:10.1371/journal.pone.0032689.g004 suggested that the calbindin 2-like protein annotated in the genome of S. purpuratus is the echinoderm orthologue of calbindin- D32k. Calbindin 2 or calretinin The BLAST search for calbindin 2 using R. norvegicus protein sequence (NP_446440.1), against the NCBI GeneBank database (Echinodermata- TaxID: 7586) showed that the most similar hits were the same as for calbindin 1, though with lower E values and higher scores: calretIso2 had an E value of 1e257 and a Score of 218, calretIso1 had an E value of 1e256 and a Score of 214, and the Sp_SCGN had an E value of 1e236 and a Score of 147. Structural comparative analysis performed between rat calbindin 2 and the sea urchin calretIso2 sequences using the ScanProsite program showed that both proteins are mainly composed of EF- hand domains, but rat calbindin 2 had only 5 domains, all of which were capable of binding Ca2+, while the sea urchin calretIso2 had 6 all of which were capable of binding Ca2+ (Fig. 5). A multiple alignment of calbindin 2 sequences, calbindin-D32k, and the calretIso2 also suggested that the latter might correspond to calbindin-D32k in the sea urchin. A phylogenetic tree from this analysis showed that calretIso2 was clustered with calbindin-D32k. These results also support our contention that the calbindin 2-like protein annotated in the genome of S. purpuratus is the echinoderm orthologue of calbindin-D32k. Calbindin-D32k The invertebrate sequence most similar to the calretIso2 protein was that of calbindin-D32k from S. kowalevskii. A Blast of calretIso2 showed that the E value and score for this protein were 2e284 and 261. Sequence alignment between these two proteins showed that they are 52% identical and 71% similar when conserved residues were included. As expected, the calbindin-D32k had a similar number of amino acids, when compared to calretIso2, 293 versus 282 amino acids, respectively. Structural analysis using the ScanProsite program showed that this protein had 6 EF-hand domains, all of which were capable of binding Ca2+ (Fig. 5). A multiple alignment of calbindin-D32k sequence in invertebrates, rat calbindin 2, and the S. purpuratus calbindin-D32k supported previous results showing a high similarity between calbindin 2 and the invertebrate calbindin-D32k, (Fig. 6a). In a phylogenetic tree done from a multiple alignment of representatives of the calbindin family (calbindin 1, calbindin 2, calbindin-D32k, and secretago- gin), two major groups were formed (Fig. 6b). The first included calbindin 1, calbindin 2, and calbindin-D32k, and the second was composed of secretagogin. Within the first cluster, two major groups were derived, one containing calbindin-D32k and a second containing calbindin 1 and 2. The sea urchin calretIso2 was grouped with the calbindin-D32k as was expected from our previous analyses. Hence, from now on we will refer to calretIso2 as S. purpuratus’ calbindin-D32k. Parvalbumin The BLAST search for parvalbumin (using R. norvegicus protein sequence- NP_071944.1- against the NCBI GeneBank database - Echinodermata: taxid: 7586) showed a predicted protein similar to calmodulin (XP_001190813.1) from the sea urchin S. purpuratus to be the most similar match. Although the e-value seems to be relatively low (3e207 and a score of 49.7), both sequences shared 33% of identities and 55% of positive residues. The structural analyses using ScanProsite between rat parvalbumin and the sea urchin predicted protein-similar to calmodulin showed that the first had two EF-hand domains, while the latter had four EF-hand domains. Interestingly, this predicted protein from sea urchin had 283 amino acids, while the rat parvalbumin had 110 amino acids. In the S. purpuratus genome database (Sea Urchin Genome Sequence Consortium 2006), three calmodulin sequences have been reported (GLEAN3_05032, GLEAN3_05033 and GLEAN3_16371). Moreover, among the higher hits from R. norvegicus Parvalbumin BLAST was calmodulin (AAY41437) from the Japanese sea cucumber Apostichopus japonicus (149 amino acids). Sequence alignment between these two proteins showed that they are 29% identical and 51% similar when positive residues were included. In summary, no sequence orthologous to the vertebrate parvalbumin was found in the echinoderms. Figure 5. Schematic domain structure of members of the calbindin subfamily in rat (R. norvegicus), fly (D. melanogaster), and sea urchin (S. purpuratus). Domains 1, 3, 4, and 5 are present in all the proteins. The second domain doesn’t bind calcium in the calbindin 1 protein, while it does in calbindin 2, and in Drosophila and S. purpuratus’ calbindin-D32k. Domain 6 is present in Drosophila calbindin-D32k and S. purpuratus calbindin-D32k, but it is only able to bind calcium in the latter. Pairwise similarities of domains between proteins were calculated by aligning the domains as predicted by ScanProsite. doi:10.1371/journal.pone.0032689.g005 PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32689