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Figure 1. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the nervous system of Holothuria glaberrima (Holothuroidea).Transverse sections through the radial nerve cords showing immunoreactivity to anti-calbindin 1 (A), anti-parvalbumin (B), and anti-calbindin 2 (C) in the ectoneural and hyponeural components of the radial nerve. Double-labeling of anti-calbindin 1 (D) and RN1 (G), anti-parvalbumin (E) and RN1 (H), and anti-calbindin 2 (F) and RN1 (I) in cells (arrows) and fibers of the connective tissue plexus of the tube feet. Co-labeling is observed with the anti-CBPs markers and RN1 in the majority of the fibers, but not in all (arrowheads). ern, ectoneural component of the radial nerve cord; hrn, hyponeural component of the radial nerve cord; *, ectoneural component cell bodies; **, hyponeural component cell bodies.
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Figure 2. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the peripheral nerve and circular muscle of Holothuria glaberrima (Holothuroidea).Longitudinal sections through the body wall showing colabeling of anti-calbindin 1 (A) and RN1 (D), anti-parvalbumin (B) and RN1 (E), and anti-calbindin 2 (C) and RN1 (F). Most of the immunoreactivity was observed in the peripheral nerves (arrowheads), while only a minor supopulations of fibers were co-labeled by the anti-CBP and RN1 (arrows).
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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.
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Figure 4. Anti-calbindin 1, anti-parvalbumin, and anti-calbindin 2 immunoreactivity in the podial nervous system of Holothuria glaberrima (Holothuroidea).Longitudinal and transverse sections through the tube feet showing immunoreactivity to anti-calbindin 1 (A,D,G,M), anti-parvalbumin (B,E,I,N), and anti-calbindin 2 (C,F,K,O) in the different subdivisions of the podial nervous system. (A–F) Immunoreactivity was observed with all markers in the podial nerve (arrows) and in the podial cylindrical fenestrated sheath (arrowheads). (G–L) Co-labeling of anti-calbindin 1 (G) and RN1 (H), anti-parvalbumin (I) and RN1 (J), and anti-calbindin 2 (K) and RN1 (L) was observed in fibers and cells (arrows) of the 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.
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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.
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Figure 6. Alignment of the peptide sequences and phylogenetic trees of members of the calbindin subfamily from different species.(A) Sequence alignment of rat calbindin 2 (NP_446440.1), Drosophila calbindin-D32k (NP_476838.1), S. kowalevskii calbindin-D32k (XP_002735965.1), and S. purpuratus calbindin-D32k proteins (XP_781517.2). Alignment of the proteins was made using the CLUSTALW multiple sequences alignment. Dots represent conserved residues and black lines below the residues represents the presence of an EF hand domain. (B) Phylogenetic tree of members of the calbindin subfamily from different species. Calb32_Dm Drosophila melanogaster (NP_476838.1), Calb32_Sk S. kowalevskii (XP_002735965.1), Calb32_Sp S. purpuratus (XP_781517.2), Calb32_Sm S. mansoni (XP_002574332.1), Calb2_Gg G. gallus (NP_990647.1), Calb2_Dr D. rerio (NP_957005.1), Calb2_Rn R. norvegicus (NP_446440.1), Calb2_Hs H. sapiens (NP_001731.2), Calb1_Xl X. laevis (NP_00108408.1), Calb1_Gg G. gallus (NP_990844.1), Calb1_Mm M. musculus (AAH16421.1), Calb1_Rn R. norvegicus (AAH81764.1), Calb1_Hs H. sapiens (NP_004920.1), SCGN_Sk S. kowalevskii (NP_001161653.1), SCGN_Sp S. purpuratus (XP_785060.2), SCGN_Dr D. rerio (NP_001005776.1), SCGN_Xl X. laevis (NP_001088097.1), SCGN_Rn R. norvegicus (NP_963855.1), SCGN_Mm M. musculus (NP_663374.1). The phylogenetic tree was constructed from an alignment created by using the unweighted pair group method with arithmetic mean. The scale bar represents 100 times the expected number of amino acid substitution.
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Figure 7. Western blot of holothurian and echinoid radial nerve cords preparations using anti-calbindin 1 and anti-parvalbumin.(A) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in H. glaberrima and L. variegatus radial nerve cords homogenates. A 28 kDa immunoreactive band corresponding to calbindin 1 was observed in the rat kidney positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain-containing protein negative control. (B) A 32 kDa immunoreactive band to anti-calbindin 1 was observed in H. glaberrima and L. variegatus radial nerve cords homogenates. A 12 kDa major immunoreactive band corresponding to calbindin 1 was observed in the rat skeletal muscle positive control and no immunoreactive bands were observed in the synthetic peptide of an EF-hand domain-containing protein negative control.
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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).
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