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

Carlos A Díaz-Balzac,Enrique M García-Rivera,Carlos I González,José E García-Arrarás,María I Lázaro-Peña,Vladimir N Uversky

doi:10.1371/journal.pone.0032689

Abstract

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.

Highlights

EF-hand domain-containing calcium-binding proteins (CBP) of the calbindin and parvalbumin subfamilies have been used during the last three decades as markers of subpopulation of neurons in the nervous system of vertebrates [1,2,3,4,5,6,7,8,9,10,11]
We identified the presence of calbindin-D32k in H. glaberrima and L. variegatus, supporting its presence in at least two classes of echinoderms Our data supports the evolutionary conservation of the calbindin subfamily, which seems to have evolved from two primitive EF-hand domain-containing CBP
In this study we report the distribution of an EF-hand domain containing CBP of the calbindin subfamily in the nervous system of the echinoderms

Summary

Introduction

EF-hand domain-containing calcium-binding proteins (CBP) of the calbindin and parvalbumin subfamilies have been used during the last three decades as markers of subpopulation of neurons in the nervous system of vertebrates [1,2,3,4,5,6,7,8,9,10,11]. The first major reports in non-vertebrates were conducted in Loligo pealeii (Lesueur, 1821) (Teuthida, Cephalopoda) [12] and Drosophila melanogaster (Meigen, 1830) (Diptera, Insecta) [13]. In the former a protein biochemically similar to calbindin 1 was characterized, though its sequence is not yet available. In the latter, the calbindin-D32k gene of Drosophila was cloned. No other CBP of the calbindin or parvalbumin subfamilies has been reported to be present in a non-vertebrate organism

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