Model for the roles of the OSTF and the carbonic anhydrase in sclerocytes of the sponge <i>S. raphanus</i>.

<p>Based on the finding that the expression of the two molecules is upregulated during Ca⁺⁺ depletion condition, it is proposed that these proteins are involved in the development of the precursor sclerocytes to the functionally active catabolic sclerocytes. OSTF forms with Cbl and Src a triple complex that stimulates the membrane-associated PI-3K and lipid metabolism. This metabolic chain is initiated by clustering integrins. These processes finally result in an increased mobility/migration of the catabolic sclerocytes and in an increased expression of the <i>carbonic anhydrase</i> gene. The latter enzyme generates protons that dissolve spicular CaCl₂ (ACC), and causes spicule resorption. Presumably occurring pH shifts within the cells are counterbalanced by a vacuolar H⁺-transporting adenosine triphosphatase and the release of Cl⁻ via the chloride/bicarbonate exchanger (see: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Pfaffl1" target="_blank">[58]</a>). In analogy to the differentiation pathway of mammalian osteoclasts it is proposed that also in sponges the differentiation of sclerocytes is under control of, hitherto unknown, differentiation factors and their receptors acting similar like OPG, RANKL and RANK in mammals.</p

Surface architecture of spicules from specimens grown in normal ambient CaCl₂ concentrations (10 mM) and cultivated in a CaCl₂-depleted environment.

<p>The isolation of the spicules has been performed after a short exposure (1 h; sheath-spicules) or after an extended exposure (5 h; purified-spicules) to NaOCl. (<b>A</b>) Sheath-spicules from specimens grown in sea water, supplemented with 10 mM CaCl₂. The surfaces of the abundantly occurring triactines (ts) are covered by organic sheaths (os). (<b>B</b> and <b>C</b>) At a higher magnification those layers, organic sheaths (os), can be resolved as circular netlike ropes whereby the individual fibrils apparently do not fuse to each other; they are tightly attached to the calcite surfaces (><). (<b>D</b> to <b>F</b>) Purified-spicules, diactines (ds) and triactines (ts), devoid of any visible sheath show a smooth surface. (<b>G</b>) Sheath-spicule from a specimen kept for 5 d in CaCl₂-depleted aqueous environment, showing likewise an organic sheath. (<b>H</b> and <b>I</b>) Purified-spicules from similarly cultivated animals; the rough surface architecture is obvious (<>). (<b>J</b>) At higher magnification the rough surface architecture can be resolved as palisade bricks, sticking out about 100 nm radially from the spicules (double-headed arrow). (<b>K</b>) The fissuring of the spicules with an almost identical depth (double-headed arrow) suggests that the calcitic material of the spicules is not homogenous with respect to their density or content in organic material. (<b>L</b>) In comparison, the smooth surface of a purified-spicule, from an animal kept at 10 mM CaCl₂, is shown.</p

Production of recombinant <i>S. raphanus</i> carbonic anhydrase (r-CA) and OSTF (r-OSTF).

<p>(<b>A</b>) Preparation of the recombinant <i>S. raphanus</i> putative carbonic anhydrase (r-CA) and analysis by NaDodSO4-PAGE and Western blotting. NaDodSO4-PAGE: (<b>M</b>) Size markers. (<b>Lane a</b>) Proteins in the bacterial pellet, obtained from induced bacteria; (<b>lane b</b>) pattern after lysis with BugBuster; (<b>lane c</b>) affinity purified r-CA. Western blotting; (<b>lane d</b>) the antiserum raised against the r-CA (PoAb-aCA) recognizes the 29-kDa recombinant protein, while (<b>lane e</b>) a pre-immune serum (p.s.) did not react. (<b>B</b>) The recombinant OSTF protein (r-OSTF). (<b>Lane a</b>) NaDodSO4-PAGE analysis of the purified protein. Western blot analysis: (<b>lane b</b>) Reactivity of the antibodies raised against OSTF (PoAb-aOSTFr) to the 25 kDa r-OSTF; while (<b>lane b</b>) the pre-immune serum (p.s.) does not react.</p

The <i>S. raphanus</i> putative carbonic anhydrase.

<p>(<b>A</b>) The sponge putative carbonic anhydrase (CA_SYCON) is aligned with the highly related sequences from the demosponge <i>S. domuncula</i>, the silicase (SIA_SUBDO; DD298191), and the carbonic anhydrases from the scleractinian <i>Acropora millepora</i> (CAr1_ACRMIL; ACJ64662.1), and the stony coral <i>Stylophora pistillata</i> (CAa_STYPI; ACA53457.1, EU159467.1), as well as with the human carbonic anhydrase 2 (CA II) (CAHB_HUMAN; O75493). The indicative sites/regions within the <i>Sycon</i> polypeptide are marked, the carbonic anhydrase alpha (vertebrate-like) group stretch (−CA−), including the His residues, functioning as Zn-binding sites, the hydrophobic parts (+hydb+), as well as the signal peptide (:signal:). Residues conserved (identical or similar) in all sequences are shown in white on black; those which share similarity to at least four residues are in black on grey. (<b>B</b>) Radial phylogenetic tree, including the mentioned sequences, together with human carbonic anhydrases of the following isoforms: I (CA-I) (CAH1_HUMAN; P00915); II (CA-II) (CAH2_HUMAN; P00918); III (CA-III) (CAH3_HUMAN; P07451); IV (CAIV_HUMAN; AAA35625.1); IV (CA-IV) (CAH4_HUMAN; P22748); VA (CAH5_HUMAN; P35218); VB (CA5B_HUMAN; CA5B_HUMAN); VI (CA-VI) (CAH6_HUMAN; P23280); VII (CA-VII) (CAH7_HUMAN; P43166); VIII (CA-VIII) (CAH8_HUMAN; P35219); IX (CA-IX) (CAH9_HUMAN; Q16790); 10 (CA-RP X) (CAHA_HUMAN; Q9NS85); XII (CA-XII) (CAHC_HUMAN; O43570); XIV (CA-XIV) (CAHE_HUMAN; Q9ULX7). In addition, the coral sequence from <i>Acropora millepora</i> (CAr2_ACRMIL; ACJ64663.1), as well as the ones from the sea anemone <i>Nematostella vectensis</i> (CAr_NEMVE; XP_001627923.1), the tunicate <i>Ciona intestinalis</i> (CA14_CIONA; XP_002123314.1); the lancelet <i>Branchiostoma floridae</i> (CAr_BRANFLO; XP_002601262.1), the shark <i>Squalus acanthias</i> (CA4_SQUAAC; AAZ03744.1); the fish <i>Oreochromis niloticus</i> (CA4_ORENI; XP_003456174.1), together with the insect enzyme from <i>D. melanogaster</i> (CAr_DROME; NP_572407.3) are included.</p

The calcareous sponge <i>Sycon raphanus</i> (Schmidt, 1862).

<p>(<b>A</b>) The species <i>S. raphanus</i> had been grouped by Haeckel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a> to the taxon <i>Sycandra</i>. Here a scheme of the morphology and the skeletal structure of <i>Sycandra hystrix</i> is given <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a>. (<b>B</b>) <i>S. raphanus</i> specimens, growing on the mussel (m) <i>Mytilus galloprovincialis</i>. The oscule of the specimens is surrounded by a pronounced corona (co), formed of spicules. On the basis of the specimens stolons/buds (st) are seen. They develop after release from the parent sponge asexually to a descendent. (<b>C</b> and <b>D</b>) Cross section through <i>S. raphanus</i> specimens, displaying the external and internal surface layer. In the center the atrium (a) is shown into which the water canals flow in. Radial aquiferous canals traverse the body that originate at the surface of the animal, via the inhalant openings (io), and end at the internal surface via exhalant pores (eo). Between the canals the mesenchyme (m) compartments is radially arranged. The slices were stained with ASTRIN. (<b>E</b>) Non-stained section through the outer part of the sponge showing the location of the two major types of spicules; (i) the diactines spicules (ds), protruding from the distal cones of the outer surface of the specimens, and (ii) the triactines (ts) that are localized within the mesohyl. The mesohyl compartment is filled with eggs/embryos (e).</p

Expression levels of the OSTF (<i>SROSTFr</i>) in <i>S. raphanus</i> in dependence on the level of Ca²⁺ in the culture medium.

<p>Expression levels of the OSTF (<i>SROSTFr</i>) in <i>S. raphanus</i> in dependence on the level of Ca²⁺ in the culture medium.</p

The <i>S. raphanus</i> putative OSTF (OSTFr_SYCON), deduced from the cDNA (<i>SROSTFr</i>).

<p>(<b>A</b>) The sponge OSTF was aligned with the related molecules from <i>S. domuncula</i> (OSTFr_SUBDO; CAJ44456.1) and human (OSTF_HOMO; EAW62571.1) as well as with the ankyrin sequence from <i>S. domuncula</i> (ANKYRIN_SUBDO ;CAH04634.1). The borders of the characteristic proline-rich region (+P+), the SH3 (∼SH3∼) as well as the domain ankyrin domain (-ANK_REP_REGION-) are marked. Residues conserved (identical or similar with respect to their physico-chemical properties) in all sequences are shown in white on black; those which share similarity between two sequences are in black on grey. (<b>B</b>) These four proteins were compared with the related polypeptide from <i>Hydra magnipapillata</i> (OSTFr_HYDRA; |XP_002165941.1), from <i>Branchiostoma floridae</i> (OSTFr_BRANFL; XP_002594542.1), from <i>Ciona intestinalis</i> (OSTFr_CIONA; XP_002126742.1), from the fish <i>Danio rerio</i> (OSTFr_DANIO gi|47086319|ref|NP_998022.1), from <i>Xenopus laevis</i> (OSTFr_XENLAE; NP_001080411.1), from the bird <i>Taeniopygia guttata</i> (OSTF_TAENGUT; XP_002190351.1), from the red deer <i>Cervus elaphus</i> (OSTFr_CERVUS; ABR68244.1), the distantly related sequences, hypothetical protein Y106G6H.14, from <i>Caenorhabditis elegans</i> (Y106G6H.14_CAEEL; NP_492738.1) and the ankyrin 2 from <i>Drosophila melanogaster</i> (OSTFr_DROME; NP_001189067.1). The tree was calculated and rooted with the plant sequence from <i>Arabidopsis thaliana</i> (ATPASE_ARATH; NP_178442.2), a proteasome non-ATPase regulatory subunit 10, as outgroup. Scale bar indicates an evolutionary distance of 0.1 aa substitutions per position in the sequence.</p

Results of the ELISA titration experiments with the phages Sycon-09 peptide, likely standing for the OSTF (Sycon-09(OSF)), and for the phage Sycon-23, indicative for the carbonic anhydrase (Sycon-23(CA)).

<p>The titer values (PFU/mL) obtained by using the antibodies against M13 revealed that the adsorption of Sycon-23(CA) and Sycon-09(OSTF) is much higher than the one read for the wild type phage M13.</p

Expression levels of the putative carbonic anhydrase (<i>SRCA</i>) gene in <i>S. raphanus</i>.

<p>The specimens were cultivated under standard condition (10 mM CaCl₂) or at Ca⁺⁺-depletion condition (1 mM CaCl₂). After incubation periods of 3 to 7 d the animals were collected, RNA prepared and the extent of gene expression was quantified by qRT-PCR, as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#s2" target="_blank">Material and Methods</a>”. Each data point represents the mRNA level of the respective expressed gene normalized to the amount of <i>β-tubulin</i> transcripts, as means ± SD (n = 6).</p