Calbindin-D32k in the Echinoderm Nervous System Figure 8. Identification of calbindin-D32k as the anti-calbindin 1 and anti-parvalbumin epitope in echinoderms. (A) Sequence alignment of H. glaberrima calbindin-D32k fragment (Calb32_Hg) and S. purpuratus calbindin-D32k isoform 1 (Calb32(1)_Sp) and isoform 2 (Calb32(2)_Sp), showing a high degree of homology. Alignment of the proteins was made using the CLUSTALW multiple sequences alignment. Dark gray represent conserved residues, light gray represent conservation of strong groups, and black lines below the residues represents the presence of an EF hand domain. (B) A 36 kDa immunoreactive band to anti-calbindin 1 and anti-parvalbumin was observed in the H. glaberrima GST-tagged calbindin-D32k fragment peptide (Calb32_Hg), and no immunoreactive band to anti-calbindin 1 and anti-parvalbumin was observed in the negative control, GST-tagged translationally controlled tumor protein (TCTP). doi:10.1371/journal.pone.0032689.g008 Anti-CBP immunoreactivity identified distinctive plexi within the connective tissue One of the most interesting findings of our study is the immunoreactivity of the CBP markers in the connective tissue plexi of the body wall and tube feet. The only other marker that has been used to identify these plexi is RN1 [27,29]. The fact that the CBP markers recognize these plexi is very interesting from a functional point of view. Trotter and Koob [41] demonstrated that calcium-dependent processes are involved in the stiffening of the body wall in the sea cucumber Cucumaria frondosa (Gunnerus, 1770) (Holothuroidea, Dendrochirotida). The importance of calcium in the connective tissue catch phenomenon has also been demon- strated due to its association with the electron-dense granules in the juxtaligamental cells [42] and its role in the hardening and softening of the intervertebral ligament of ophiruoids [43]. Therefore, the echinoderms EF-hand containing CBPs are excellent candidates to act as calcium buffers and might play some important role in the control of the mutable connective tissue, which still needs to be defined and further investigated. Anti-calbindin 1 and anti-parvalbumin recognize the same antigen, echinoderms’ calbindin-D32k Bioinformatics analysis of members of the calbindin subfamily in the genome of the sea urchin S. purpuratus were performed to further single out which subfamily member is/are our markers recognizing. We focused on the presence of EF-hand domain- containing CBPs of the calbindin and parvalbumin subfamily, as these molecules are the ones recognized by these markers in vertebrates. Using this approach, we identified two proteins that are part of the calbindin subfamily of CBP, a putative calbindin 2 and secretagogin, but no proteins of the parvalbumin subfamily. Both of these proteins are predicted gene models that were formulated from the whole genome-sequencing project [17]. Thus we had two possibilities as to which antigen is/are the CBP marker recognizing in echinoderms. Although the labeling of antigens by antibodies provides some information on the molecular structure of the antigen, in no way this provides definite proof of the antigen identity. Thus, additional studies were performed to better define the type(s) of CBP(s) of the calbindin subfamily being recognized by the antibodies in echinoderms. Western blot analyses to the three markers showed 32 kDa immunoreactive bands for anti-calbindin 1 and anti-parvalbumin. This observation, together with the similar labeling pattern observed in immunohistochemistry, raised the question as to whether these antibodies are recognizing the same antigen in H. glaberrima. This was certainly a possibility, especially considering that the location of fibers and cells labeled by immunohistochemistry is very similar for both. The location of the immunoreactivity within the immunopositive structures is the same, but the number of fibers and cells is not. This difference can be explained by a difference in the affinity of the antibodies for the PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e32689