Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata)

Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.