Calbindin-D32k in the Echinoderm Nervous System Figure 3. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the body wall nervous system of Holothuria glaberrima (Holothuroidea). Longitudinal sections through the body wall showing immunoreactivity to anti-calbindin 1 (A,E,I), anti-parvalbumin (B,F,J), and anti-calbindin 2 (C,G,K) as compared to RN1 (D,H,L) immunoreactivity in the different layers of the body wall. Immunoreactivity of the internal connective tissue plexus (A–D), external connective tissue (E–H) and epidermis (I–L) showed that the anti-CBP only labeled a minor subpopulation of fibers and cells within these plexi, as compared to RN1 which labeled a large subpopulations of cells and fibers of the body wall nervous system. doi:10.1371/journal.pone.0032689.g003 antibodies in the echinoderm nervous system, we performed the following analyses: (1) an orthologous search for the respective protein sequence from Rattus norvegicus using the NCBI’s GeneBank database (Echinodermata; TaxID: 7586) and the S. purpuratus annotated genome in SpBase and (2) structural comparison of the EF-hand domains of related proteins. If an orthologue sequence was obtained, multiple sequences alignments were performed. Calbindin 1 or calbindin-D28k A BLAST of rat calbindin 1 (AAH81764.1) against the NCBI Echinodermata sequences was performed to identify the presence of orthologous proteins in the echinoderms. Rat calbindin 1 sequence was used since this was the immunogen against which the anti-calbindin 1 antibody was made. The best hit obtained in this search was a predicted protein known as ‘‘similar to ENSANGP00000013966 isoform 2’’ (XP_781517.2), which cor- responded to the S. purpuratus putative calbindin 2, as annotated by Kathy Foltz [17], with an E value of 1e250 and a Score of 196 (from now on CalretIso2 or Sp_Calb32). The second best hit was the isoform 1 of the same predicted protein (XP_001178717.1) (from now on CalretIso1) with an E value of 4e250 and a Score of 194; while the third best hit was a hypothetical protein (XP_785060.2), which corresponds to the S. purpuratus secretago- gin, as annotated by Kathy Foltz [17], (from now on Sp_SCGN), with an E value of 3e231 and a Score of 131. A comparison performed between both calbindin 2 isoforms identified in S. purpuratus sequences revealed two slight differences between them. First, isoform 1 had nine more amino acids than isoform 2 (residues 204 to 212); and second, they differed in five amino acids (residues 198 to 203). An alignment between calretIso2 from S. purpuratus and calbindin 1 from R. norvegicus showed 42% of identities and 60% of positives. Nonetheless, these two sequences differed in length and molecular weight. In rats, like in other vertebrates, calbindin 1 has 261 amino acids with a molecular weight of 28 kDa. Meanwhile, the sea urchin calretIso2 had 288 amino acids with a predicted molecular weight of 32 kDa. Structural comparative analysis performed between rat calbindin 1, rat calbindin 2, Drosophila calbindin-D32k and the sea urchin calbindin-D32k sequences using the ScanProsite program showed that all proteins had different numbers of EF-hand domains. Rat calbindin 1 and 2 had only 5 EF-hand domains, of which 4 and 5 of these are capable of binding Ca2+ respectively, while Drosophila and the S. purpuratus calbindin-D32k had 6, of which 5 and 6 of these are capable of binding Ca2+ respectively (Fig. 5). Furthermore, a BLAST of calretIso2 against GeneBank showed that the most similar sequence was a predicted protein similar to calbindin-D32k (XP_002735965.1) of the hemichordate Saccoglos- sus kowalevskii (Agassiz, 1873) (Enteropneusta, Hemichordata). The second best hit was another predicted protein similar to calbindin- D32k (XP_975683.1) from the invertebrate Tribolium castaneum (Herbst, 1797) (Insecta, Arthropoda). A multiple alignment of the best hits of calbindin 1 sequences (-D28k and -D32k) with 11 species (5 from vertebrates and 4 from invertebrate), suggested that calretIso2 might correspond to calbindin-D32k in the sea urchin. A phylogenetic tree from this analysis showed that Sp_Calb32 clustered with the calbindin-D32k. All together, these results PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32689